Antisense oligomers for treatment of conditions and diseases

ABSTRACT

Alternative splicing events in SCN1A gene can lead to non-productive mRNA transcripts which in turn can lead to aberrant protein expression, and therapeutic agents which can target the alternative splicing events in SCN1A gene can modulate the expression level of functional proteins in Dravet Syndrome patients and/or inhibit aberrant protein expression. Such therapeutic agents can be used to treat a condition caused by SCN1A, SCN8A or SCN5A protein deficiency.

CROSS-REFERENCE

This application is a continuation of international patent applicationno. PCT/US2018/48031 filed on Aug. 24, 2018 which claims the benefit ofU.S. Provisional Application No. 62/550,462, filed on Aug. 25, 2017,U.S. Provisional Application No. 62/575,901, filed on Oct. 23, 2017,U.S. Provisional Application No. 62/667,356, filed on May 4, 2018, U.S.Provisional Application No. 62/671,745, filed on May 15, 2018, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 6, 2020, isnamed 47991-719_601_SL.txt and is 998,937 bytes in size.

BACKGROUND OF THE INVENTION

Nervous system disorders are often associated with channelopathy,characterized by the disturbed function of ion channels that mediateneuronal excitability, neuronal interactions, and brain functions atlarge. Mutations in the SCN1A gene, which is part of theSCN1A-SCN2A-SCN3A gene cluster that encodes alpha-pore forming subunitsof the neuronal voltage gated sodium channel, are associated withdevelopment of disease number of diseases and conditions, such as DravetSyndrome (DS) (Miller, et al., 1993-2015, GeneReviews, Eds. Pagon R A,et al. Seattle (Wash.): University of Washington, Seattle, Bookshelf ID:NBK1318, and Mulley, et al., 2005, Hum. Mutat. 25: 535-542).

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, is a method of modulatingexpression of SCN1A protein in a cell having an mRNA that contains anon-sense mediated RNA decay-inducing exon (NMD exon mRNA) and encodesSCN1A protein, the method comprising contacting a therapeutic agent tothe cell, whereby the therapeutic agent modulates splicing of the NMDexon from the NMD exon mRNA encoding SCN1A protein, thereby modulatingthe level of processed mRNA encoding SCN1A protein, and modulatingexpression of SCN1A protein in the cell. In some embodiments, thetherapeutic agent (a) binds to a targeted portion of the NMD exon mRNAencoding SCN1A; (b) modulates binding of a factor involved in splicingof the NMD exon mRNA; or (c) a combination of (a) and (b). In someembodiments, the therapeutic agent interferes with binding of the factorinvolved in splicing of the NMD exon from a region of the targetedportion. In some embodiments, the targeted portion is proximal to theNMD exon. In some embodiments, the targeted portion is at most about1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400nucleotides, about 300 nucleotides, about 200 nucleotides, about 100nucleotides, about 80 nucleotides, about 70 nucleotides, about 60nucleotides, about 50 nucleotides upstream of 5′ end of the NMD exon. Insome embodiments, the targeted portion is at least about 1500nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700nucleotides, about 600 nucleotides, about 500 nucleotides, about 400nucleotides, about 300 nucleotides, about 200 nucleotides, about 100nucleotides, about 80 nucleotides, about 70 nucleotides, about 60nucleotides, about 50 nucleotides, about 40 nucleotides, about 30nucleotides, about 20 nucleotides, about 10 nucleotides, about 5nucleotides, about 4 nucleotides, about 2 nucleotides, about 1nucleotides upstream of 5′ end of the NMD exon. In some embodiments, thetargeted portion is at most about 1500 nucleotides, about 1000nucleotides, about 800 nucleotides, about 700 nucleotides, about 600nucleotides, about 500 nucleotides, about 400 nucleotides, about 300nucleotides, about 200 nucleotides, about 100 nucleotides, about 80nucleotides, about 70 nucleotides, about 60 nucleotides, about 50nucleotides downstream of 3′ end of the NMD exon. In some embodiments,the targeted portion is at least about 1500 nucleotides, about 1000nucleotides, about 800 nucleotides, about 700 nucleotides, about 600nucleotides, about 500 nucleotides, about 400 nucleotides, about 300nucleotides, about 200 nucleotides, about 100 nucleotides, about 80nucleotides, about 70 nucleotides, about 60 nucleotides, about 50nucleotides, about 40 nucleotides, about 30 nucleotides, about 20nucleotides, about 10 nucleotides, about 5 nucleotides, about 4nucleotides, about 2 nucleotides, about 1 nucleotides downstream of 3′end of the NMD exon. In some embodiments, the targeted portion islocated in an intronic region between two canonical exonic regions ofthe NMD exon mRNA encoding SCN1A, and wherein the intronic regioncontains the NMD exon. In some embodiments, the targeted portion atleast partially overlaps with the NMD exon. In some embodiments, thetargeted portion at least partially overlaps with an intron upstream ofthe NMD exon. In some embodiments, the targeted portion comprises 5′ NMDexon-intron junction or 3′ NMD exon-intron junction. In someembodiments, the targeted portion is within the NMD exon. In someembodiments, the targeted portion comprises about 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, or more consecutive nucleotides of the NMD exon. In someembodiments, the NMD exon mRNA encoding SCN1A comprises a sequence withat least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to anyone of SEQ ID NOs: 2 or 7-10. In some embodiments, the NMD exon mRNAencoding SCN1A is encoded by a genetic sequence with at least about 80%,85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NOs: 1 or 3-6.In some embodiments, the targeted portion is at most about 1500nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700nucleotides, about 600 nucleotides, about 500 nucleotides, about 400nucleotides, about 300 nucleotides, about 200 nucleotides, about 100nucleotides, about 80 nucleotides, about 70 nucleotides, about 60nucleotides, about 50 nucleotides upstream of genomic site GRCh37/hg19:chr2:166,863,803. In some embodiments, the targeted portion is about1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300nucleotides, about 200 nucleotides, about 100 nucleotides, about 80nucleotides, about 70 nucleotides, about 60 nucleotides, about 50nucleotides, about 40 nucleotides, about 30 nucleotides, about 20nucleotides, about 10 nucleotides, about 5 nucleotides, about 4nucleotides, about 2 nucleotides, about 1 nucleotides upstream ofgenomic site GRCh37/hg19: chr2:166,863,803. In some embodiments, thetargeted portion is at most about 1500 nucleotides, about 1000nucleotides, about 800 nucleotides, about 700 nucleotides, about 600nucleotides, about 500 nucleotides, about 400 nucleotides, about 300nucleotides, about 200 nucleotides, about 100 nucleotides, about 80nucleotides, about 70 nucleotides, about 60 nucleotides, about 50nucleotides downstream of genomic site GRCh37/hg19: chr2:166,863,740. Insome embodiments, the targeted portion is about 1000 nucleotides, about800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500nucleotides, about 400 nucleotides, about 300 nucleotides, about 200nucleotides, about 100 nucleotides, about 80 nucleotides, about 70nucleotides, about 60 nucleotides, about 50 nucleotides, about 40nucleotides, about 30 nucleotides, about 20 nucleotides, about 10nucleotides, about 5 nucleotides, about 4 nucleotides, about 2nucleotides, about 1 nucleotides downstream of genomic site GRCh37/hg19:chr2:166,863,740. In some embodiments, the targeted portion of the NMDexon mRNA encoding SCN1A comprises a sequence with at least 80%, 85%,90%, 95%, 97%, or 100% sequence identity to a region comprising at least8 contiguous nucleic acids of SEQ ID NO: SEQ ID NOs: 2 or 7-10. In someembodiments, the therapeutic agent is an antisense oligomer (ASO) andwherein the ASO comprises a sequence that is at least about 80%, 85%,90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 21-67,210-256, or 304-379. In some embodiments, the targeted portion of theNMD exon mRNA encoding SCN1A is within the non-sense mediated RNAdecay-inducing exon 20x of SCN1A. In some embodiments, the therapeuticagent is an antisense oligomer (ASO) and wherein the ASO comprises asequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100%identity to any one of SEQ ID NOs: 42-50, or 231-239. In someembodiments, the targeted portion of the NMD exon mRNA encoding SCN1A isupstream or downstream of the non-sense mediated RNA decay-inducing exon20x of SCN1A. In some embodiments, the therapeutic agent is an antisenseoligomer (ASO) and wherein the ASO comprises a sequence that is at leastabout 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ IDNOs: 21-38, 53-67, 210-227, or 242-256. In some embodiments, thetargeted portion of the NMD exon mRNA comprises an exon-intron junctionof exon 20x of SCN1A. In some embodiments, the therapeutic agent is anantisense oligomer (ASO) and wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any oneof SEQ ID NOs: 39-41, 51, 52, 228-230, 240, or 241. In some embodiments,the therapeutic agent promotes exclusion of the NMD exon from theprocessed mRNA encoding SCN1A protein. In some embodiments, exclusion ofthe NMD exon from the processed mRNA encoding SCN1A protein in the cellcontacted with the therapeutic agent is increased about 1.1 to about10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 toabout 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold,about 4 to about 8-fold, about 4 to about 9-fold, at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about4-fold, at least about 5-fold, or at least about 10-fold, compared toexclusion of the NMD exon from the processed mRNA encoding SCN1A proteinin a control cell. In some embodiments, the therapeutic agent increaseslevel of the processed mRNA encoding SCN1A protein in the cell. In someembodiments, an amount of the processed mRNA encoding SCN1A protein inthe cell contacted with the therapeutic agent is increased about 1.1 toabout 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold,about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold,about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 toabout 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, atleast about 1.1-fold, at least about 1.5-fold, at least about 2-fold, atleast about 2.5-fold, at least about 3-fold, at least about 3.5-fold, atleast about 4-fold, at least about 5-fold, or at least about 10-fold,compared to an total amount of the processed mRNA encoding SCN1A proteinin a control cell. In some embodiments, the therapeutic agent increasesexpression of SCN1A protein in the cell. In some embodiments, an amountof SCN1A produced in the cell contacted with the therapeutic agent isincreased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold,about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 toabout 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold,about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about2-fold, at least about 2.5-fold, at least about 3-fold, at least about3.5-fold, at least about 4-fold, at least about 5-fold, or at leastabout 10-fold, compared to an total amount of SCN1A produced in acontrol cell. In some embodiments, the therapeutic agent inhibitsexclusion of the NMD exon from the processed mRNA encoding SCN1Aprotein. In some embodiments, exclusion of the NMD exon from theprocessed mRNA encoding SCN1A protein in the cell contacted with thetherapeutic agent is decreased about 1.1 to about 10-fold, about 1.5 toabout 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold,about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 toabout 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 2.5-fold, at leastabout 3-fold, at least about 3.5-fold, at least about 4-fold, at leastabout 5-fold, or at least about 10-fold, compared to exclusion of theNMD exon from the processed mRNA encoding SCN1A protein in a controlcell. In some embodiments, the therapeutic agent decreases level of theprocessed mRNA encoding SCN1A protein in the cell. In some embodiments,an amount of the processed mRNA encoding SCN1A protein in the cellcontacted with the therapeutic agent is decreased about 1.1 to about10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 toabout 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold,about 4 to about 8-fold, about 4 to about 9-fold, at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about4-fold, at least about 5-fold, or at least about 10-fold, compared to antotal amount of the processed mRNA encoding SCN1A protein in a controlcell. In some embodiments, the therapeutic agent decreases expression ofSCN1A protein in the cell. In some embodiments, an amount of SCN1Aproduced in the cell contacted with the therapeutic agent is decreasedabout 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold,about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 toabout 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, atleast about 1.1-fold, at least about 1.5-fold, at least about 2-fold, atleast about 2.5-fold, at least about 3-fold, at least about 3.5-fold, atleast about 4-fold, at least about 5-fold, or at least about 10-fold,compared to an total amount of SCN1A produced in a control cell. In someembodiments, the therapeutic agent is an antisense oligomer (ASO) andwherein the antisense oligomer comprises a backbone modificationcomprising a phosphorothioate linkage or a phosphorodiamidate linkage.In some embodiments, the therapeutic agent is an antisense oligomer(ASO) and wherein the antisense oligomer comprises a phosphorodiamidatemorpholino, a locked nucleic acid, a peptide nucleic acid, a2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety. In someembodiments, the therapeutic agent is an antisense oligomer (ASO) andwherein the antisense oligomer comprises at least one modified sugarmoiety. In some embodiments, each sugar moiety is a modified sugarmoiety. In some embodiments, the therapeutic agent is an antisenseoligomer (ASO) and wherein the antisense oligomer consists of from 8 to50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20nucleobases, or 12 to 15 nucleobases. In some embodiments, thetherapeutic agent is an antisense oligomer (ASO) and wherein theantisense oligomer is at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, complementary to the targetedportion of the NMD exon mRNA encoding the protein. In some embodiments,the method further comprises assessing SCN1A mRNA or protein expression.In some embodiments, the cells are ex vivo.

Disclosed herein, in certain embodiments, is a method of treating adisease or condition in a subject in need thereof by modulatingexpression of SCN1A protein in a cell of the subject, comprising:contacting the cell of the subject with a therapeutic agent thatmodulates splicing of a non-sense mediated mRNA decay-inducing exon (NMDexon) from an mRNA in the cell that contains the NMD exon and encodesSCN1A, thereby modulating the level of processed mRNA encoding the SCN1Aprotein, and modulating expression of SCN1A protein in the cell of thesubject. In some embodiments, the therapeutic agent (a) binds to atargeted portion of the NMD exon mRNA encoding SCN1A; (b) modulatesbinding of a factor involved in splicing of the NMD exon mRNA; or (c) acombination of (a) and (b). In some embodiments, the therapeutic agentinterferes with binding of the factor involved in splicing of the NMDexon from a region of the targeted portion. In some embodiments, thetargeted portion is proximal to the NMD exon. In some embodiments, thetargeted portion is at most about 1500 nucleotides, about 1000nucleotides, about 800 nucleotides, about 700 nucleotides, about 600nucleotides, about 500 nucleotides, about 400 nucleotides, about 300nucleotides, about 200 nucleotides, about 100 nucleotides, about 80nucleotides, about 70 nucleotides, about 60 nucleotides, about 50nucleotides upstream of 5′ end of the NMD exon. In some embodiments, thetargeted portion is at least about 1500 nucleotides, about 1000nucleotides, about 800 nucleotides, about 700 nucleotides, about 600nucleotides, about 500 nucleotides, about 400 nucleotides, about 300nucleotides, about 200 nucleotides, about 100 nucleotides, about 80nucleotides, about 70 nucleotides, about 60 nucleotides, about 50nucleotides, about 40 nucleotides, about 30 nucleotides, about 20nucleotides, about 10 nucleotides, about 5 nucleotides, about 4nucleotides, about 2 nucleotides, about 1 nucleotides upstream of 5′ endof the NMD exon. In some embodiments, the targeted portion is at mostabout 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides,about 700 nucleotides, about 600 nucleotides, about 500 nucleotides,about 400 nucleotides, about 300 nucleotides, about 200 nucleotides,about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about60 nucleotides, about 50 nucleotides downstream of 3′ end of the NMDexon. In some embodiments, the targeted portion is at least about 1500nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700nucleotides, about 600 nucleotides, about 500 nucleotides, about 400nucleotides, about 300 nucleotides, about 200 nucleotides, about 100nucleotides, about 80 nucleotides, about 70 nucleotides, about 60nucleotides, about 50 nucleotides, about 40 nucleotides, about 30nucleotides, about 20 nucleotides, about 10 nucleotides, about 5nucleotides, about 4 nucleotides, about 2 nucleotides, about 1nucleotides downstream of 3′ end of the NMD exon. In some embodiments,the targeted portion is located in an intronic region between twocanonical exonic regions of the NMD exon mRNA encoding SCN1A, andwherein the intronic region contains the NMD exon. In some embodiments,the targeted portion at least partially overlaps with the NMD exon. Insome embodiments, the targeted portion at least partially overlaps withan intron upstream of the NMD exon. In some embodiments, the targetedportion comprises 5′ NMD exon-intron junction or 3′ NMD exon-intronjunction. In some embodiments, the targeted portion is within the NMDexon. In some embodiments, the targeted portion comprises about 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or more consecutive nucleotides of the NMD exon. Insome embodiments, the NMD exon mRNA encoding SCN1A comprises a sequencewith at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identityto any one of SEQ ID NOs: 2 or 7-10. In some embodiments, the NMD exonmRNA encoding SCN1A is encoded by a genetic sequence with at least about80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NOs: 1 or3-6. In some embodiments, the targeted portion is at most about 1500nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700nucleotides, about 600 nucleotides, about 500 nucleotides, about 400nucleotides, about 300 nucleotides, about 200 nucleotides, about 100nucleotides, about 80 nucleotides, about 70 nucleotides, about 60nucleotides, about 50 nucleotides upstream of genomic site GRCh37/hg19:chr2:166,863,803. In some embodiments, the targeted portion is about1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300nucleotides, about 200 nucleotides, about 100 nucleotides, about 80nucleotides, about 70 nucleotides, about 60 nucleotides, about 50nucleotides, about 40 nucleotides, about 30 nucleotides, about 20nucleotides, about 10 nucleotides, about 5 nucleotides, about 4nucleotides, about 2 nucleotides, about 1 nucleotides upstream ofgenomic site GRCh37/hg19: chr2:166,863,803. In some embodiments, thetargeted portion is at most about 1500 nucleotides, about 1000nucleotides, about 800 nucleotides, about 700 nucleotides, about 600nucleotides, about 500 nucleotides, about 400 nucleotides, about 300nucleotides, about 200 nucleotides, about 100 nucleotides, about 80nucleotides, about 70 nucleotides, about 60 nucleotides, about 50nucleotides downstream of genomic site GRCh37/hg19: chr2:166,863,740. Insome embodiments, the targeted portion is about 1000 nucleotides, about800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500nucleotides, about 400 nucleotides, about 300 nucleotides, about 200nucleotides, about 100 nucleotides, about 80 nucleotides, about 70nucleotides, about 60 nucleotides, about 50 nucleotides, about 40nucleotides, about 30 nucleotides, about 20 nucleotides, about 10nucleotides, about 5 nucleotides, about 4 nucleotides, about 2nucleotides, about 1 nucleotides downstream of genomic site GRCh37/hg19:chr2:166,863,740. In some embodiments, the targeted portion of the NMDexon mRNA encoding SCN1A comprises a sequence with at least 80%, 85%,90%, 95%, 97%, or 100% sequence identity to a region comprising at least8 contiguous nucleic acids of SEQ ID NO: SEQ ID NOs: 2 or 7-10. In someembodiments, the therapeutic agent is an antisense oligomer (ASO) andwherein the ASO comprises a sequence that is at least about 80%, 85%,90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 21-67,210-256, or 304-379. In some embodiments, the targeted portion of theNMD exon mRNA encoding SCN1A is within the non-sense mediated RNAdecay-inducing exon 20x of SCN1A. In some embodiments, the therapeuticagent is an antisense oligomer (ASO) and wherein the ASO comprises asequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100%identity to any one of SEQ ID NOs: 42-50, or 231-239. In someembodiments, the targeted portion of the NMD exon mRNA encoding SCN1A isupstream or downstream of the non-sense mediated RNA decay-inducing exon20x of SCN1A. In some embodiments, the therapeutic agent is an antisenseoligomer (ASO) and wherein the ASO comprises a sequence that is at leastabout 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ IDNOs: 21-38, 53-67, 210-227, or 242-256. In some embodiments, thetargeted portion of the NMD exon mRNA comprises an exon-intron junctionof exon 20x of SCN1A. In some embodiments, the therapeutic agent is anantisense oligomer (ASO) and wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any oneof SEQ ID NOs: 39-41, 51, 52, 228-230, 240, or 241. In some embodiments,the therapeutic agent promotes exclusion of the NMD exon from theprocessed mRNA encoding SCN1A protein. In some embodiments, exclusion ofthe NMD exon from the processed mRNA encoding SCN1A protein in the cellcontacted with the therapeutic agent is increased about 1.1 to about10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 toabout 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold,about 4 to about 8-fold, about 4 to about 9-fold, at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about4-fold, at least about 5-fold, or at least about 10-fold, compared toexclusion of the NMD exon from the processed mRNA encoding SCN1A proteinin a control cell. In some embodiments, the therapeutic agent increaseslevel of the processed mRNA encoding SCN1A protein in the cell. In someembodiments, an amount of the processed mRNA encoding SCN1A protein inthe cell contacted with the therapeutic agent is increased about 1.1 toabout 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold,about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold,about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 toabout 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, atleast about 1.1-fold, at least about 1.5-fold, at least about 2-fold, atleast about 2.5-fold, at least about 3-fold, at least about 3.5-fold, atleast about 4-fold, at least about 5-fold, or at least about 10-fold,compared to an total amount of the processed mRNA encoding SCN1A proteinin a control cell. In some embodiments, the therapeutic agent increasesexpression of SCN1A protein in the cell. In some embodiments, an amountof SCN1A produced in the cell contacted with the therapeutic agent isincreased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold,about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 toabout 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold,about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about2-fold, at least about 2.5-fold, at least about 3-fold, at least about3.5-fold, at least about 4-fold, at least about 5-fold, or at leastabout 10-fold, compared to an total amount of SCN1A produced in acontrol cell. In some embodiments, the therapeutic agent inhibitsexclusion of the NMD exon from the processed mRNA encoding SCN1Aprotein. In some embodiments, exclusion of the NMD exon from theprocessed mRNA encoding SCN1A protein in the cell contacted with thetherapeutic agent is decreased about 1.1 to about 10-fold, about 1.5 toabout 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold,about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 toabout 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 2.5-fold, at leastabout 3-fold, at least about 3.5-fold, at least about 4-fold, at leastabout 5-fold, or at least about 10-fold, compared to exclusion of theNMD exon from the processed mRNA encoding SCN1A protein in a controlcell. In some embodiments, the therapeutic agent decreases level of theprocessed mRNA encoding SCN1A protein in the cell. In some embodiments,an amount of the processed mRNA encoding SCN1A protein in the cellcontacted with the therapeutic agent is decreased about 1.1 to about10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 toabout 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold,about 4 to about 8-fold, about 4 to about 9-fold, at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about4-fold, at least about 5-fold, or at least about 10-fold, compared to antotal amount of the processed mRNA encoding SCN1A protein in a controlcell. In some embodiments, the therapeutic agent decreases expression ofSCN1A protein in the cell. In some embodiments, an amount of SCN1Aproduced in the cell contacted with the therapeutic agent is decreasedabout 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold,about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 toabout 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, atleast about 1.1-fold, at least about 1.5-fold, at least about 2-fold, atleast about 2.5-fold, at least about 3-fold, at least about 3.5-fold, atleast about 4-fold, at least about 5-fold, or at least about 10-fold,compared to an total amount of SCN1A produced in a control cell. In someembodiments, the therapeutic agent is an antisense oligomer (ASO) andwherein the antisense oligomer comprises a backbone modificationcomprising a phosphorothioate linkage or a phosphorodiamidate linkage.In some embodiments, the therapeutic agent is an antisense oligomer(ASO) and wherein the antisense oligomer comprises a phosphorodiamidatemorpholino, a locked nucleic acid, a peptide nucleic acid, a2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety. In someembodiments, the therapeutic agent is an antisense oligomer (ASO) andwherein the antisense oligomer comprises at least one modified sugarmoiety. In some embodiments, each sugar moiety is a modified sugarmoiety. In some embodiments, the therapeutic agent is an antisenseoligomer (ASO) and wherein the antisense oligomer consists of from 8 to50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20nucleobases, or 12 to 15 nucleobases. In some embodiments, thetherapeutic agent is an antisense oligomer (ASO) and wherein theantisense oligomer is at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or 100%, complementary to the targetedportion of the NMD exon mRNA encoding the protein. In some embodiments,the method further comprises assessing SCN1A mRNA or protein expression.In some embodiments, the disease or condition is induced by aloss-of-function mutation in Na_(v)1.1. In some embodiments, the diseaseor condition is associated with haploinsufficiency of the SCN1A gene,and wherein the subject has a first allele encoding a functional SCN1A,and a second allele from which SCN1A is not produced or produced at areduced level, or a second allele encoding a nonfunctional SCN1A or apartially functional SCN1A. In some embodiments, the disease orcondition is encephalopathy. In some embodiments, the encephalopathy isepileptic encephalopathy. In some embodiments, the disease or conditionis Dravet Syndrome (DS); severe myoclonic epilepsy of infancy(SMEI)-borderland (SMEB); Febrile seizure (FS); epilepsy, generalized,with febrile seizures plus (GEFS+); epileptic encephalopathy, earlyinfantile, 13; cryptogenic generalized epilepsy; cryptogenic focalepilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; Westsyndrome; idiopathic spasms; early myoclonic encephalopathy; progressivemyoclonic epilepsy; alternating hemiplegia of childhood; unclassifiedepileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP);sick sinus syndrome 1; autism; or malignant migrating partial seizuresof infancy. In some embodiments, GEFS+ is epilepsy, generalized, withfebrile seizures plus, type 2. In some embodiments, the Febrile seizureis Febrile seizures, familial, 3A. In some embodiments, SMEB is SMEBwithout generalized spike wave (SMEB-SW), SMEB without myoclonicseizures (SMEB-M), SMEB lacking more than one feature of SMEI (SMEB-O),or intractable childhood epilepsy with generalized tonic-clonic seizures(ICEGTC). In some embodiments, the therapeutic agent promotes exclusionof the NMD exon from the processed mRNA encoding SCN1A protein andincreases the expression of SCN1A in the cell. In some embodiments, thetherapeutic agent is an antisense oligomer (ASO) and wherein the ASOcomprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or100% complimentary to any one of SEQ ID NOs: 22-24, 26, 27, 29-35,37-62, 64-67, or 304-379. In some embodiments, the disease or conditionis induced by a gain-of-function mutation in Na_(v)1.1. In someembodiments, the subject has an allele from which SCN1A is produced atan increased level, or an allele encoding a mutant SCN1A that inducesincreased activity of Na_(v)1.1 in the cell. In some embodiments, thedisease or condition is migraine. In some embodiments, the migraine ismigraine, familial hemiplegic, 3. In some embodiments, the disease orcondition is a Na_(v)1.1 genetic epilepsy. In some embodiments, thetherapeutic agent inhibits exclusion of the NMD exon from the processedmRNA encoding SCN1A protein and decreases the expression of SCN1A in thecell. In some embodiments, the therapeutic agent is an antisenseoligomer (ASO) and wherein the ASO comprises a sequence that is at leastabout 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQID NOs: 21, 25, 28, 36, or 63. In some embodiments, the subject is ahuman. In some embodiments, the subject is a non-human animal. In someembodiments, the subject is a fetus, an embryo, or a child. In someembodiments, the therapeutic agent is administered by intrathecalinjection, intracerebroventricular injection, intraperitoneal injection,intramuscular injection, subcutaneous injection, intravitreal, orintravenous injection of the subject. In some embodiments, the methodfurther comprises administering a second therapeutic agent to thesubject. In some embodiments, the second therapeutic agent is a smallmolecule. In some embodiments, the second therapeutic agent is an ASO.In some embodiments, the ASO comprises a sequence that is at least about80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs:115-161. In some embodiments, the second therapeutic agent correctsintron retention. In some embodiments, the disease or condition isAlzheimer's Disease, SCN2A encephalopathy, SCN8A encephalopathy, orSCN5A arrhythmia. In some embodiments, the disease or condition isAlzheimer's Disease, SCN2A encephalopathy, SCN8A encephalopathy, orSCN5A arrythmia. In some embodiments, the cells are ex vivo.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-C depict a schematic representation of a target mRNA thatcontains a non-sense mediated RNA decay-inducing exon (NMD exon mRNA)and therapeutic agent-mediated exclusion of the nonsense-mediated mRNAdecay-inducing exon to increase expression of the full-length targetprotein or functional RNA. FIG. 1A shows a cell divided into nuclear andcytoplasmic compartments. In the nucleus, a pre-mRNA transcript of atarget gene undergoes splicing to generate mRNA, and this mRNA isexported to the cytoplasm and translated into target protein. For thistarget gene, some fraction of the mRNA contains a nonsense-mediated mRNAdecay-inducing exon (NMD exon mRNA) that is degraded in the cytoplasm,thus leading to no target protein production. FIG. 1B shows an exampleof the same cell divided into nuclear and cytoplasmic compartments.Treatment with a therapeutic agent, such as an antisense oligomer (ASO),promotes the exclusion of the nonsense-mediated mRNA decay-inducing exonand results in an increase in mRNA, which is in turn translated intohigher levels of target protein. FIG. 1C is a schematic representationof therapeutic ASO-mediated exclusion of a nonsense-mediated mRNAdecay-inducing exon, which turns a non-productive mRNA into a productivemRNA and increases expression of the full-length target protein from theproductive mRNA.

FIG. 2 depicts identification of an exemplary nonsense-mediated mRNAdecay (NMD)-inducing exon in the SCN1A gene. The identification of theNMD-inducing exon in the SCN1A gene using comparative genomics is shown,visualized in the UCSC genome browser. The upper panel shows a graphicrepresentation of the SCN1A gene to scale. The conservation level across100 vertebrate species is shown as peaks. The highest peaks correspondto exons (black boxes), while no peaks are observed for the majority ofthe introns (lines with arrow heads). Peaks of conservation wereidentified in intron 20 (NM_006920), shown in the middle panel.Inspection of the conserved sequences identified an exon-like sequenceof 64 bp (bottom panel, sequence highlighted in grey) flanked by 3′ and5′ splice sites (underlined sequence). Inclusion of this exon leads to aframeshift and the introduction of a premature termination codon in exon21 rendering the transcript a target of NMD.

FIG. 3A depicts confirmation of NMD-inducing exon via cycloheximidetreatment. RT-PCR analysis using cytoplasmic RNA from DMSO-treated(CHX−) or cycloheximide-treated (CHX+) Neuro 2A (mouse neural progenitorcells) and primers in exon 21 and a downstream exon confirmed thepresence of a band corresponding to the NMD-inducing exon (21x). Theidentity of the product was confirmed by sequencing. Densitometryanalysis of the bands was performed to calculate percent exon 21xinclusion of total SCN1A transcript. Treatment of Neuro 2A withcycloheximide (CHX+) to inhibit NMD led to a 2-fold increase of theproduct corresponding to the NMD-inducing exon 21x in the cytoplasmicfraction (cf. light grey bar, CHX−, to dark grey bar, CHX+).

FIG. 3B depicts confirmation of NMD-inducing exon via cycloheximidetreatment. RT-PCR analysis using cytoplasmic RNA from DMSO-treated(CHX−) or cycloheximide-treated (CHX+) RenCell VM (human neuralprogenitor cells) and primers in exon 20 and exon 23 confirmed thepresence of a band corresponding to the NMD-inducing exon (20x). Theidentity of the product was confirmed by sequencing. Densitometryanalysis of the bands was performed to calculate percent exon 20xinclusion of total SCN1A transcript. Treatment of RenCell VM withcycloheximide (CHX+) to inhibit NMD led to a 2-fold increase of theproduct corresponding to the NMD-inducing exon 20x in the cytoplasmicfraction (cf. light grey bar, CHX−, to dark grey bar, CHX+).

FIG. 4 depicts an exemplary SCN1A exon 20x region ASO walk. A graphicrepresentation of an ASO walk performed for SCN1A exon 20x regiontargeting sequences upstream of the 3′ splice site, across the 3′splicesite, exon 20x, across the 5′ splice site, and downstream of the 5′splice site using 2′-MOE ASOs, PS backbone, is shown. ASOs were designedto cover these regions by shifting 5 nucleotides at a time.

FIG. 5A depicts SCN1A exon 20x region ASO walk evaluated by RT-PCR. Arepresentative PAGE shows SYBR-safe-stained RT-PCR products of SCN1Amock-treated (Sham), SMN-control ASO treated (SMN), or treated with a2′-MOE ASO targeting the exon 20x region as described herein in theExamples and in the description of FIG. 4, at 20 μM concentration inRenCell VM cells by gymnotic uptake. Two products corresponding to exon20x inclusion (top band) and full-length (exon 20x exclusion, bottomband) were quantified.

FIG. 5B depicts a graph plotting the percent exon 20x inclusion from thedata in FIG. 5A. The black line indicates no change with respect toSham.

FIG. 5C depicts a graph of the full-length products normalized to RPL32internal control and the fold-change relative to Sham is plotted. Theblack line indicates a ratio of 1 and no change with respect to Sham.

FIG. 6 depicts an exemplary SCN1A exon 20x region ASO walk evaluated byRT-qPCR. SYBR-green RT-qPCR SCN1A amplification results normalized toRPL32, obtained using the same ASO uptake experiment that were evaluatedby SYBR-safe RT-PCR as shown in FIG. 5, are plotted as fold changerelative to Sham confirming the SYBR-safe RT-PCR results. The black lineindicates a ratio of 1 (no change with respect to sham).

FIG. 7A depicts a table with members of the sodium voltage-gaitedchannel alpha subunit members. Arrows correspond to bar colors in FIG.7B. X denotes no expression detected.

FIG. 7B depicts selected ASOs evaluated by Taqman qPCR of SCN1A, SCN2A,SCN3A, SCN8A, and SCN9A to assess target selectivity. Taqman-qPCRamplification results normalized to RPL32, obtained using Ex20x+1,IVS20x+18, and IVS20x+33 ASOs, are plotted as fold change relative toSham. The black line indicates a ratio of 1 (no change with respect tosham).

FIG. 8A depicts exemplary dose-dependent effect of selected ASO inCXH-treated cells. A representative PAGE showing SYBR-safe-stainedRT-PCR products of mouse Scn1a mock-treated (Sham, RNAiMAX alone), ortreated with Ex21x+1 2′-MOE ASO targeting the exon 21x (mousenomenclature, corresponds to human exon 20x), at 30 nM, 80 nM, and 200nM concentrations in Neuro 2A (mouse neuroblastoma) cells by RNAiMAXtransfection is shown. Ex21x+1 (mouse nomenclature) and Ex20x+1 (humannomenclature) are identical. Two products corresponding to exon 20xinclusion (top band) and full-length (exon 20x exclusion, bottom band)were quantified.

FIG. 8B depicts a graph plotting the percent exon 20x inclusion from thedata in FIG. 7A. The black line indicates no change with respect toSham.

FIG. 8C depicts an exemplary graph of the full-length productsnormalized to Hprt internal control and fold-change relative to Sham areplotted. The black line indicates a ratio of 1 and no change withrespect to Sham.

FIG. 9A depicts exemplary results from intravitreal (IVT) injection ofselected ASOs in C57BL6J mice (male, 3 months old). PAGE gels ofSYBR-safe-stained RT-PCR products of mouse Scn1a from PBS-injected (1μL) left eye (−) or IVS20x-21, Ex21x+1, IVS21x+18, IVS21x+33 or Cep290(negative control ASO; Gerard et al, Mol. Ther. Nuc. Ac., 2015) 2′-MOEASO-injected (1 μL) right eye (+) at 10 mM concentration are shown.Ex21x+1, IVS21x+18, and IVS21x+33 (mouse nomenclature) and Ex20x+1,IVS20x+18, and IVS20x+33 (human nomenclature) are identical. Twoproducts corresponding to exon 21x inclusion (top band) and full-length(exon 21x exclusion, bottom band) were quantified.

FIG. 9B depicts a graph plotting the percent exon 21x inclusion from thedata in FIG. 9A. White bars correspond to ASO-injected eyes and greybars correspond to PBS-injected eyes, n=5 in each group.

FIG. 9C depicts a graph of the full-length products were normalized toGapdh internal control and fold-change of ASO-injected eye relative toPBS-injected eye is plotted. The black line indicates a ratio of 1 andno change with respect to PBS, n=5 in each group.

FIG. 10A depicts exemplary results from intracerebroventricular (ICV)injection of selected ASOs in C57BL6J mice (male, 3 months old). PAGEgels of SYBR-safe-stained RT-PCR products of mouse Scn1a from uninjected(-, no ASO control), or 300 μg of Cep290 (negative control ASO; Gerardet al, Mol. Ther. Nuc. Ac., 2015), Ex21x+1, IVS21x+18, IVS21x+33 2′-MOEASO-injected brains are shown. Ex21x+1, IVS21x+18, and IVS21x+33 (mousenomenclature) and Ex20x+1, IVS20x+18, and IVS20x+33 (human nomenclature)are identical. Two products corresponding to exon 21x inclusion (topband) and full-length (exon 21x exclusion, bottom band) were quantified.

FIG. 10B depicts a graph plotting the percent exon 21x inclusion fromthe data in FIG. 10A, n=6 (each targeting ASO), n=5 (Cep290 ASO), n=1(uninjected, no ASO control).

FIG. 10C depicts a graph from results of a Taqman qPCR assay performedusing two different probes spanning exons 21 and 22 junction. Theproducts were normalized to Gapdh internal control and fold-change ofASO-injected relative to Cep290-injected brains is plotted. The blackline indicates a ratio of 1 and no change with respect to Cep290, n=6(each targeting ASO), n=5 (Cep290 ASO), n=1 (uninjected, no ASOcontrol).

FIG. 11A depicts exemplary results from intracerebroventricular (ICV)injection of selected ASOs in C57BL6J mice (male, 3 months old). PAGEgels of SYBR-safe-stained RT-PCR products of mouse Scn1a from 300 ug ofCep290 (negative control ASO; Gerard et al, Mol. Ther. Nuc. Ac., 2015),or 33 ug, 100 ug, and 300 ug of Ex21x+1 2′-MOE ASO-injected brains.Ex21x+1 (mouse nomenclature) and Ex20x+1, (human nomenclature) areidentical. Two products corresponding to exon 21x inclusion (top band)and full-length (exon 21x exclusion, bottom band) were quantified.

FIG. 11B depicts a graph plotting the percent exon 21x inclusion fromthe data in FIG. 11A, n=5 (each group).

FIG. 11C depicts a graph from results of a Taqman qPCR assay performedusing two different probes spanning exons 21 and 22 junction. Theproducts were normalized to Gapdh internal control and fold-change ofASO-injected relative to Cep290-injected brains is plotted. The blackline indicates a ratio of 1 and no change with respect to Cep290, n=5(each group).

FIG. 12A depicts exemplary results from intracerebroventricular (ICV)injection of a selected ASO in C57BL6J mice (postnatal day 2). PAGE gelsof SYBR-safe-stained RT-PCR products of mouse Scn1a from uninjected (-,no ASO control), or 20 μg Ex21x+1 2′-MOE ASO-injected brains are shown.Two products corresponding to exon 21x inclusion (top band) andfull-length (exon 21x exclusion, bottom band) were quantified. Ex21x+1(mouse nomenclature) and Ex20x+1 (human nomenclature) are identical.

FIG. 12B depicts a graph plotting the percent exon 21x inclusion fromthe data in FIG. 12A, n=4 (each group).

FIG. 12C depicts a graph from results of a Taqman qPCR assay performedusing two different probes spanning exons 21 and 22 junction. Theproducts were normalized to Gapdh internal control and fold-change ofASO-injected relative to no-ASO-control brains is plotted. The blackline indicates a ratio of 1 and no change with respect to no-ASOcontrol, n=4 (each group).

FIG. 13A depicts a graph plotting the percent exon 21x inclusion in theindicated mouse CNS samples.

FIG. 13B depicts a graph plotting the percent exon 20x inclusion in theindicated human CNS samples.

FIG. 14A depicts a graph plotting the percent decrease in exon 21xinclusion at the indicated doses.

FIG. 14B depicts a graph plotting the percent increase in Scn1a mRNA atthe indicated doses.

FIG. 14C depicts a graph plotting the percent increase in Nav 1.1protein levels at the indicated doses.

FIG. 15A depicts a graph plotting the percent decrease in exon 21xinclusion at the indicated doses.

FIG. 15B depicts a graph plotting the percent increase in Scn1a mRNA atthe indicated doses.

FIG. 16 depicts a selected Scn1a targeting ASO administered at a bugdose via ICV injection in postnatal day 2 mice evaluated at day 5post-injection by Taqman qPCR of SCN1A, SCN2A, SCN3A, SCN4A, SCN5A,SCN7A, SCN8A, SCN9A, SCN10A, and SCN11A to assess target selectivity.Taqman-qPCR amplification results normalized to Gapdh, obtained usingEx20x+1 ASO, are plotted as fold change relative to PBS injected mice.

FIGS. 17A and 17B depict exemplary results from intracerebroventricular(ICV) injection at postnatal day 2 of a selected ASO at the indicateddose in wild type (WT) or heterozygous Dravet mice (HET) F1 mice from129S-Scn1a^(tm1Kea)×C57BL/6J crosses at 3 days post-injection.

FIG. 17A depicts a graph from results of a Taqman qPCR assay performedusing a probe spanning exons 21 and 22. The products were normalized toGapdh internal control and fold-change of ASO-injected relative toPBS-injected brains is plotted.

FIG. 17B depicts a graph from results of a western blot performed usingan anti-Nav1.1 antibody. The products were normalized to Ponceau-stainedbands and fold-change of ASO-injected relative to PBS-injected brains isplotted.

FIG. 18 depicts exemplary results of a SCN1A exon 20x region ASOmicrowalk in RenCells via free uptake. ASOs were designed to coverregions around three previously identified targeting ASOs in FIG. 6(marked by stars) by shifting 1 nucleotides at a time (6-41) or bydecreasing the length of ASO 17 (1-5). The graph depicts the percentexon 20x inclusion as measured by SYBR-green qPCR. The black lineindicates no change with respect to no ASO (−).

FIG. 19 is a graph plotting increase in Scn1a mRNA level in coronalbrain slices of mice over the time post injection of a SCN1A targetingASO. As depicted, increase in Scn1a mRNA level was maintained for atleast 80 days post-injection.

FIG. 20 is an exemplary survival curve demonstrating 100% survivalbenefit provided by a SCN1A targeting ASO in Dravet mouse model. +/+stands for WT genotype, and +/− stands for 129S-scn1a^(tm1Kea)heterozygous genotype (Dravet mouse model); A stands for PBS treatment,and B stands for ASO treatment. As depicted, mice in A +/− group (Dravetmice receiving PBS treatment) started to die from about postnatal day16, whereas all mice of other three groups, including B+/−(Drave micereceiving ASO treatment) group, survived through at least postnatal day35.

DETAILED DESCRIPTION OF THE INVENTION

Splicing and Nonsense-Mediated mRNA Decay

Intervening sequences or introns are removed by a large and highlydynamic RNA-protein complex termed the spliceosome, which orchestratescomplex interactions between primary transcripts, small nuclear RNAs(snRNAs) and a large number of proteins. Spliceosomes assemble ad hoc oneach intron in an ordered manner, starting with recognition of the 5′splice site (5′ss) by U1 snRNA or the 3′splice site (3′ss) by the U2pathway, which involves binding of the U2 auxiliary factor (U2AF) to the3′ss region to facilitate U2 binding to the branch point sequence (BPS).U2AF is a stable heterodimer composed of a U2AF2-encoded 65-kD subunit(U2AF65), which binds the polypyrimidine tract (PPT), and aU2AF1-encoded 35-kD subunit (U2AF35), which interacts with highlyconserved AG dinucleotides at 3′ss and stabilizes U2AF65 binding. Inaddition to the BPS/PPT unit and 3′ss/5′ss, accurate splicing requiresauxiliary sequences or structures that activate or repress splice siterecognition, known as intronic or exonic splicing enhancers orsilencers. These elements allow genuine splice sites to be recognizedamong a vast excess of cryptic or pseudo-sites in the genome of highereukaryotes, which have the same sequences but outnumber authentic sitesby an order of magnitude. Although they often have a regulatoryfunction, the exact mechanisms of their activation or repression arepoorly understood.

The decision of whether to splice or not to splice can be typicallymodeled as a stochastic rather than deterministic process, such thateven the most defined splicing signals can sometimes splice incorrectly.However, under normal conditions, pre-mRNA splicing proceeds atsurprisingly high fidelity. This is attributed in part to the activityof adjacent cis-acting auxiliary exonic and intronic splicing regulatoryelements (ESRs or ISRs). Typically, these functional elements areclassified as either exonic or intronic splicing enhancers (ESEs orISEs) or silencers (ESSs or ISSs) based on their ability to stimulate orinhibit splicing, respectively. Although there is now evidence that someauxiliary cis-acting elements may act by influencing the kinetics ofspliceosome assembly, such as the arrangement of the complex between U1snRNP and the 5′ss, it seems very likely that many elements function inconcert with trans-acting RNA-binding proteins (RBPs). For example, theserine- and arginine-rich family of RBPs (SR proteins) is a conservedfamily of proteins that have a key role in defining exons. SR proteinspromote exon recognition by recruiting components of the pre-spliceosometo adjacent splice sites or by antagonizing the effects of ESSs in thevicinity. The repressive effects of ESSs can be mediated by members ofthe heterogeneous nuclear ribonucleoprotein (hnRNP) family and can alterrecruitment of core splicing factors to adjacent splice sites. Inaddition to their roles in splicing regulation, silencer elements aresuggested to have a role in repression of pseudo-exons, sets of decoyintronic splice sites with the typical spacing of an exon but without afunctional open reading frame. ESEs and ESSs, in cooperation with theircognate trans-acting RBPs, represent important components in a set ofsplicing controls that specify how, where and when mRNAs are assembledfrom their precursors.

The sequences marking the exon-intron boundaries are degenerate signalsof varying strengths that can occur at high frequency within humangenes. In multi-exon genes, different pairs of splice sites can belinked together in many different combinations, creating a diverse arrayof transcripts from a single gene. This is commonly referred to asalternative pre-mRNA splicing. Although most mRNA isoforms produced byalternative splicing can be exported from the nucleus and translatedinto functional polypeptides, different mRNA isoforms from a single genecan vary greatly in their translation efficiency. Those mRNA isoformswith premature termination codons (PTCs) at least 50 bp upstream of anexon junction complex are likely to be targeted for degradation by thenonsense-mediated mRNA decay (NMD) pathway. Mutations in traditional(BPS/PPT/3′ss/5′ss) and auxiliary splicing motifs can cause aberrantsplicing, such as exon skipping or cryptic (or pseudo-) exon inclusionor splice-site activation, and contribute significantly to humanmorbidity and mortality. Both aberrant and alternative splicing patternscan be influenced by natural DNA variants in exons and introns.

Given that exon-intron boundaries can occur at any of the threepositions of a codon, it is clear that only a subset of alternativesplicing events can maintain the canonical open reading frame. Forexample, only exons that are evenly divisible by 3 can be skipped orincluded in the mRNA without any alteration of reading frame. Splicingevents that do not have compatible phases will induce a frame-shift.Unless reversed by downstream events, frame-shifts can certainly lead toone or more PTCs, probably resulting in subsequent degradation by NMD.NMD is a translation-coupled mechanism that eliminates mRNAs containingPTCs. NMD can function as a surveillance pathway that exists in alleukaryotes. NMD can reduce errors in gene expression by eliminating mRNAtranscripts that contain premature stop codons. Translation of theseaberrant mRNAs could, in some cases, lead to deleteriousgain-of-function or dominant-negative activity of the resultingproteins. NMD targets not only transcripts with PTCs but also a broadarray of mRNA isoforms expressed from many endogenous genes, suggestingthat NMD is a master regulator that drives both fine and coarseadjustments in steady-state RNA levels in the cell.

A NMD-inducing exon (NIE) is an exon or a pseudo-exon that is a regionwithin an intron and can activate the NMD pathway if included in amature RNA transcript. In the constitutive splicing events, the introncontaining an NIE is usually spliced out, but the intron or a portionthereof (e.g. NIE) can be retained during alternative or aberrantsplicing events. Mature mRNA transcripts containing such an NIE can benon-productive due to frame shift which induce NMD pathway. Inclusion ofa NIE in mature RNA transcripts can downregulate gene expression. mRNAtranscripts containing an NIE can be referred as “NIE containing mRNA”or “NMD exon mRNA” in the current disclosure.

Cryptic (or pseudo-splice sites) have the same splicing recognitionsequences as genuine splice sites but are not used in the splicingreactions. They outnumber genuine splice sites in the human genome by anorder of a magnitude and are normally repressed by thus far poorlyunderstood molecular mechanisms. Cryptic 5′ splice sites have theconsensus NNN/GUNNNN or NNN/GCNNNN where N is any nucleotide and/is theexon-intron boundary. Cryptic 3′ splice sites have the consensus NAG/N.Their activation is positively influenced by surrounding nucleotidesthat make them more similar to the optimal consensus of authentic splicesites, namely MAG/GURAGU and YAG/G, respectively, where M is C or A, Ris G or A, and Y is C or U.

Splice sites and their regulatory sequences can be readily identified bya skilled person using suitable algorithms publicly available, listedfor example in Kralovicova, J. and Vorechovsky, I. (2007) Global controlof aberrant splice site activation by auxiliary splicing sequences:evidence for a gradient in exon and intron definition. Nucleic AcidsRes., 35, 6399-6413,(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095810/pdf/gkm680.pdf)

The cryptic splice sites or splicing regulatory sequences may competefor RNA-binding proteins such as U2AF with a splice site of the NIE. Inone embodiment, an agent may bind to the cryptic splice site or splicingregulatory sequences to prevent the binding of RNA-binding proteins andthereby favoring utilization of the NIE splice sites.

In one embodiment, the cryptic splice site may not comprise the 5′ or 3′splice site of the NIE. The cryptic splice site may be at least 10nucleotides upstream of the NIE 5′ splice site. The cryptic splice sitemay be at least 20 nucleotides upstream of the NIE 5′ splice site. Thecryptic splice site may be at least 50 nucleotides upstream of the NIE5′ splice site. The cryptic splice site may be at least 100 nucleotidesupstream of the NIE 5′ splice site. The cryptic splice site may be atleast 200 nucleotides upstream of the NIE 5′ splice site.

The cryptic splice site may be at least 10 nucleotides downstream of theNIE 3′ splice site. The cryptic splice site may be at least 20nucleotides downstream of the NIE 3′ splice site. The cryptic splicesite may be at least 50 nucleotides downstream of the NIE 3′ splicesite. The cryptic splice site may be at least 100 nucleotides downstreamof the NIE 3′ splice site. The cryptic splice site may be at least 200nucleotides downstream of the NIE 3′ splice site.

Target Transcripts

In some embodiments, the methods of the present disclosure exploit thepresence of NIE in the pre-mRNA transcribed from the SCN1A gene.Splicing of the identified SCN1A NIE pre-mRNA species to producefunctional mature SCN1A mRNA can be induced using a therapeutic agentsuch as an ASO that stimulates exon skipping of an NIE. Induction ofexon skipping can result in inhibition of an NMD pathway. The resultingmature SCN1A mRNA can be translated normally without activating NMDpathway, thereby increasing the amount of SCN1A protein in the patient'scells and alleviating symptoms of a condition associated with SCN1Adeficiency, such as Dravet Syndrome (DS); Epilepsy, generalized, withfebrile seizures plus, type 2; Febrile seizures, familial, 3A; Autism;Epileptic encephalopathy, early infantile, 13; Sick sinus syndrome 1;Alzheimer's disease; or SUDEP.

In various embodiments, the present disclosure provides a therapeuticagent which can target SCN1A mRNA transcripts to modulate, e.g., enhanceor inhibit, splicing or protein expression level. The therapeutic agentcan be a small molecule, polynucleotide, or polypeptide. In someembodiments, the therapeutic agent is an ASO. Various regions orsequences on the SCN1A pre-mRNA can be targeted by a therapeutic agent,such as an ASO. In some embodiments, the ASO targets a SCN1A pre-mRNAtranscript containing an NIE. In some embodiments, the ASO targets asequence within an NIE of a SCN1A pre-mRNA transcript. In someembodiments, the ASO targets a sequence upstream (or 5′) from the 5′ endof an NIE (3′ss) of a SCN1A pre-mRNA transcript. In some embodiments,the ASO targets a sequence downstream (or 3′) from the 3′ end of an NIE(5′ss) of a SCN1A pre-mRNA transcript. In some embodiments, the ASOtargets a sequence that is within an intron flanking on the 5′ end ofthe NIE of a SCN1A pre-mRNA transcript. In some embodiments, the ASOtargets a sequence that is within an intron flanking the 3′ end of theNIE of a SCN1A pre-mRNA transcript. In some embodiments, the ASO targetsa sequence comprising an NIE-intron boundary of a SCN1A pre-mRNAtranscript. An NIE-intron boundary can refer to the junction of anintron sequence and an NIE region. The intron sequence can flank the 5′end of the NIE, or the 3′ end of the NIE. In some embodiments, the ASOtargets a sequence within an exon of a SCN1A pre-mRNA transcript. Insome embodiments, the ASO targets a sequence within an intron of a SCN1Apre-mRNA transcript. In some embodiments, the ASO targets a sequencecomprising both a portion of an intron and a portion of an exon.

In some embodiments, a therapeutic agent described herein modulatesbinding of a factor involved in splicing of the NMD exon mRNA.

In some embodiments, a therapeutic agent described herein interfereswith binding of a factor involved in splicing of the NMD exon mRNA.

In some embodiments, a therapeutic agent described herein preventsbinding of a factor involved in splicing of the NMD exon mRNA.

In some embodiments, a therapeutic agent targets a targeted portionlocated in an intronic region between two canonical exonic regions ofthe NMD exon mRNA encoding SCN1A, and wherein the intronic regioncontains the NMD exon.

In some embodiments, a therapeutic agent targets a targeted portion atleast partially overlaps with the NMD exon.

In some embodiments, a therapeutic agent targets a targeted portion thatis at least partially overlaps with an intron upstream of the NMD exon.

In some embodiments, a therapeutic agent targets a targeted portionwithin the NMD exon.

In some embodiments, a therapeutic agent targets a targeted portioncomprising at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutivenucleotides of the NMD exon. In some embodiments, a therapeutic agenttargets a targeted portion comprising at most about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, or more consecutive nucleotides of the NMD exon. In someembodiments, a therapeutic agent targets a targeted portion comprisingabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of theNMD exon.

In some embodiments, a therapeutic agent targets a targeted portionproximal to the NMD exon.

In some embodiments, the ASO targets a sequence from about 4 to about300 nucleotides upstream (or 5′) from the 5′ end of the NIE. In someembodiments, the ASO targets a sequence from about 1 to about 20nucleotides, about 20 to about 50 nucleotides, about 50 to about 100nucleotides, about 100 to about 150 nucleotides, about 150 to about 200nucleotides, about 200 to about 250 nucleotides, about 250 to about 300,about 250 to about 300 nucleotides, about 350 to about 400 nucleotides,about 450 to about 500 nucleotides, about 550 to about 600 nucleotides,about 650 to about 700 nucleotides, about 750 to about 800 nucleotides,about 850 to about 900 nucleotides, about 950 to about 1000 nucleotides,about 1050 to about 1100 nucleotides, about 1150 to about 1200nucleotides, about 1250 to about 1300 nucleotides, about 1350 to about1400 nucleotides, or about 1450 to about 1500 nucleotides upstream (or5′) from the 5′ end of the NIE region. In some embodiments, the ASO maytarget a sequence more than 300 nucleotides upstream from the 5′ end ofthe NIE. In some embodiments, the ASO targets a sequence from about 4 toabout 300 nucleotides downstream (or 3′) from the 3′ end of the NIE. Insome embodiments, the ASO targets a sequence about 1 to about 20nucleotides, about 20 to about 50 nucleotides, about 50 to about 100nucleotides, about 100 to about 150 nucleotides, about 150 to about 200nucleotides, about 200 to about 250 nucleotides, about 250 to about 300nucleotides, about 350 to about 400 nucleotides, about 450 to about 500nucleotides, about 550 to about 600 nucleotides, about 650 to about 700nucleotides, about 750 to about 800 nucleotides, about 850 to about 900nucleotides, about 950 to about 1000 nucleotides, about 1050 to about1100 nucleotides, about 1150 to about 1200 nucleotides, about 1250 toabout 1300 nucleotides, about 1350 to about 1400 nucleotides, or about1450 to about 1500 nucleotides downstream from the 3′ end of the NIE. Insome embodiments, the ASO targets a sequence more than 300 nucleotidesdownstream from the 3′ end of the NIE.

In some embodiments, the ASO targets a sequence from about 4 to about300 nucleotides upstream (or 5′) from the 5′ end of the NIE. In someembodiments, the ASO targets a sequence at least about 1 nucleotide, atleast about 10 nucleotides, at least about 20 nucleotides, at leastabout 50 nucleotides, at least about 80 nucleotides, at least about 85nucleotides, at least about 90 nucleotides, at least about 95nucleotides, at least about 96 nucleotides, at least about 97nucleotides, at least about 98 nucleotides, at least about 99nucleotides, at least about 100 nucleotides, at least about 101nucleotides, at least about 102 nucleotides, at least about 103nucleotides, at least about 104 nucleotides, at least about 105nucleotides, at least about 110 nucleotides, at least about 120nucleotides, at least about 150 nucleotides, at least about 200nucleotides, at least about 300 nucleotides, at least about 400nucleotides, at least about 500 nucleotides, at least about 600nucleotides, at least about 700 nucleotides, at least about 800nucleotides, at least about 900 nucleotides, or at least about 1000nucleotides upstream (or 5′) from the 5′ end of the NIE region. In someembodiments, the ASO targets a sequence about 4 to about 300 nucleotidesdownstream (or 3′) from the 3′ end of the NIE. In some embodiments, theASO targets a sequence at least about 1 nucleotide, at least about 10nucleotides, at least about 20 nucleotides, at least about 50nucleotides, at least about 80 nucleotides, at least about 85nucleotides, at least about 90 nucleotides, at least about 95nucleotides, at least about 96 nucleotides, at least about 97nucleotides, at least about 98 nucleotides, at least about 99nucleotides, at least about 100 nucleotides, at least about 101nucleotides, at least about 102 nucleotides, at least about 103nucleotides, at least about 104 nucleotides, at least about 105nucleotides, at least about 110 nucleotides, at least about 120nucleotides, at least about 150 nucleotides, at least about 200nucleotides, at least about 300 nucleotides, at least about 400nucleotides, at least about 500 nucleotides, at least about 600nucleotides, at least about 700 nucleotides, at least about 800nucleotides, at least about 900 nucleotides, or at least about 1000nucleotides downstream from the 3′ end of the NIE. In some embodiments,the ASO targets a sequence more than 300 nucleotides downstream from the3′ end of the NIE.

In some embodiments, the ASO targets a sequence from about 4 to about300 nucleotides upstream (or 5′) from the 5′ end of the NIE. In someembodiments, the ASO targets a sequence at most about 10 nucleotides, atmost about 20 nucleotides, at most about 50 nucleotides, at most about80 nucleotides, at most about 85 nucleotides, at most about 90nucleotides, at most about 95 nucleotides, at most about 96 nucleotides,at most about 97 nucleotides, at most about 98 nucleotides, at mostabout 99 nucleotides, at most about 100 nucleotides, at most about 101nucleotides, at most about 102 nucleotides, at most about 103nucleotides, at most about 104 nucleotides, at most about 105nucleotides, at most about 110 nucleotides, at most about 120nucleotides, at most about 150 nucleotides, at most about 200nucleotides, at most about 300 nucleotides, at most about 400nucleotides, at most about 500 nucleotides, at most about 600nucleotides, at most about 700 nucleotides, at most about 800nucleotides, at most about 900 nucleotides, at most about 1000nucleotides, at most about 1100 nucleotides, at most about 1200nucleotides, at most about 1300 nucleotides, at most about 1400nucleotides, or at most about 1500 nucleotides upstream (or 5′) from the5′ end of the NIE region. In some embodiments, the ASO targets asequence about 4 to about 300 nucleotides downstream (or 3′) from the 3′end of the NIE. In some embodiments, the ASO targets a sequence at mostabout 10 nucleotides, at most about 20 nucleotides, at most about 50nucleotides, at most about 80 nucleotides, at most about 85 nucleotides,at most about 90 nucleotides, at most about 95 nucleotides, at mostabout 96 nucleotides, at most about 97 nucleotides, at most about 98nucleotides, at most about 99 nucleotides, at most about 100nucleotides, at most about 101 nucleotides, at most about 102nucleotides, at most about 103 nucleotides, at most about 104nucleotides, at most about 105 nucleotides, at most about 110nucleotides, at most about 120 nucleotides, at most about 150nucleotides, at most about 200 nucleotides, at most about 300nucleotides, at most about 400 nucleotides, at most about 500nucleotides, at most about 600 nucleotides, at most about 700nucleotides, at most about 800 nucleotides, at most about 900nucleotides, or at most about 1000 nucleotides, at most about 1100nucleotides, at most about 1200 nucleotides, at most about 1300nucleotides, at most about 1400 nucleotides, or at most about 1500nucleotides downstream from the 3′ end of the NIE. In some embodiments,the ASO targets a sequence more than 300 nucleotides downstream from the3′ end of the NIE.

In some embodiments, the NIE as described herein is located betweenGRCh37/hg19: chr2:166,863,740 and GRCh37/hg19: chr2:166,863,803, asdepicted in FIG. 2. In some embodiments, the 5′ end of the NIE islocated at GRCh37/hg19: chr2:166,863,803. In some embodiments, the 3′end of the NIE is located at GRCh37/hg19: chr2:166,863,740.

In some embodiments, In some embodiments, the ASO targets a sequencefrom about 4 to about 300 nucleotides upstream (or 5′) from genomic siteGRCh37/hg19: chr2:166,863,803. In some embodiments, the ASO targets asequence about 1 to about 20 nucleotides, about 20 to about 50nucleotides, about 50 to about 100 nucleotides, about 100 to about 150nucleotides, about 150 to about 200 nucleotides, about 200 to about 250nucleotides, about 250 to about 300, about 250 to about 300 nucleotides,about 350 to about 400 nucleotides, about 450 to about 500 nucleotides,about 550 to about 600 nucleotides, about 650 to about 700 nucleotides,about 750 to about 800 nucleotides, about 850 to about 900 nucleotides,about 950 to about 1000 nucleotides, about 1050 to about 1100nucleotides, about 1150 to about 1200 nucleotides, about 1250 to about1300 nucleotides, about 1350 to about 1400 nucleotides, or about 1450 toabout 1500 nucleotides upstream (or 5′) from genomic site GRCh37/hg19:chr2:166,863,803. In some embodiments, the ASO may target a sequencemore than 300 nucleotides upstream from genomic site GRCh37/hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence fromabout 4 to about 300 nucleotides downstream (or 3′) from GRCh37/hg19:chr2:166,863,740. In some embodiments, the ASO targets a sequence about1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 toabout 100 nucleotides, about 100 to about 150 nucleotides, about 150 toabout 200 nucleotides, about 200 to about 250 nucleotides, about 250 toabout 300 nucleotides, about 350 to about 400 nucleotides, about 450 toabout 500 nucleotides, about 550 to about 600 nucleotides, about 650 toabout 700 nucleotides, about 750 to about 800 nucleotides, about 850 toabout 900 nucleotides, about 950 to about 1000 nucleotides, about 1050to about 1100 nucleotides, about 1150 to about 1200 nucleotides, about1250 to about 1300 nucleotides, about 1350 to about 1400 nucleotides, orabout 1450 to about 1500 nucleotides downstream from GRCh37/hg19:chr2:166,863,740. In some embodiments, the ASO targets a sequence morethan 300 nucleotides downstream from GRCh37/hg19: chr2: 166,863,740.

In some embodiments, the ASO targets a sequence from about 4 to about300 nucleotides upstream (or 5′) from genomic site GRCh37/hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence atleast about 1 nucleotide, at least about 10 nucleotides, at least about20 nucleotides, at least about 50 nucleotides, at least about 80nucleotides, at least about 85 nucleotides, at least about 90nucleotides, at least about 95 nucleotides, at least about 96nucleotides, at least about 97 nucleotides, at least about 98nucleotides, at least about 99 nucleotides, at least about 100nucleotides, at least about 101 nucleotides, at least about 102nucleotides, at least about 103 nucleotides, at least about 104nucleotides, at least about 105 nucleotides, at least about 110nucleotides, at least about 120 nucleotides, at least about 150nucleotides, at least about 200 nucleotides, at least about 300nucleotides, at least about 400 nucleotides, at least about 500nucleotides, at least about 600 nucleotides, at least about 700nucleotides, at least about 800 nucleotides, at least about 900nucleotides, or at least about 1000 nucleotides upstream (or 5′) fromgenomic site GRCh37/hg19: chr2:166,863,803. In some embodiments, the ASOtargets a sequence from about 4 to about 300 nucleotides downstream (or3′) from GRCh37/hg19: chr2:166,863,740. In some embodiments, the ASOtargets a sequence at least about 1 nucleotide, at least about 10nucleotides, at least about 20 nucleotides, at least about 50nucleotides, at least about 80 nucleotides, at least about 85nucleotides, at least about 90 nucleotides, at least about 95nucleotides, at least about 96 nucleotides, at least about 97nucleotides, at least about 98 nucleotides, at least about 99nucleotides, at least about 100 nucleotides, at least about 101nucleotides, at least about 102 nucleotides, at least about 103nucleotides, at least about 104 nucleotides, at least about 105nucleotides, at least about 110 nucleotides, at least about 120nucleotides, at least about 150 nucleotides, at least about 200nucleotides, at least about 300 nucleotides, at least about 400nucleotides, at least about 500 nucleotides, at least about 600nucleotides, at least about 700 nucleotides, at least about 800nucleotides, at least about 900 nucleotides, or at least about 1000nucleotides downstream from GRCh37/hg19: chr2:166,863,740. In someembodiments, the ASO targets a sequence more than 300 nucleotidesdownstream from GRCh37/hg19: chr2:166,863,740.

In some embodiments, the ASO targets a sequence from about 4 to about300 nucleotides upstream (or 5′) from genomic site GRCh37/hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence atmost about 10 nucleotides, at most about 20 nucleotides, at most about50 nucleotides, at most about 80 nucleotides, at most about 85nucleotides, at most about 90 nucleotides, at most about 95 nucleotides,at most about 96 nucleotides, at most about 97 nucleotides, at mostabout 98 nucleotides, at most about 99 nucleotides, at most about 100nucleotides, at most about 101 nucleotides, at most about 102nucleotides, at most about 103 nucleotides, at most about 104nucleotides, at most about 105 nucleotides, at most about 110nucleotides, at most about 120 nucleotides, at most about 150nucleotides, at most about 200 nucleotides, at most about 300nucleotides, at most about 400 nucleotides, at most about 500nucleotides, at most about 600 nucleotides, at most about 700nucleotides, at most about 800 nucleotides, at most about 900nucleotides, at most about 1000 nucleotides, at most about 1100nucleotides, at most about 1200 nucleotides, at most about 1300nucleotides, at most about 1400 nucleotides, or at most about 1500nucleotides upstream (or 5′) from genomic site GRCh37/hg19:chr2:166,863,803. In some embodiments, the ASO targets a sequence fromabout 4 to about 300 nucleotides downstream (or 3′) from GRCh37/hg19:chr2:166,863,740. In some embodiments, the ASO targets a sequence atmost about 10 nucleotides, at most about 20 nucleotides, at most about50 nucleotides, at most about 80 nucleotides, at most about 85nucleotides, at most about 90 nucleotides, at most about 95 nucleotides,at most about 96 nucleotides, at most about 97 nucleotides, at mostabout 98 nucleotides, at most about 99 nucleotides, at most about 100nucleotides, at most about 101 nucleotides, at most about 102nucleotides, at most about 103 nucleotides, at most about 104nucleotides, at most about 105 nucleotides, at most about 110nucleotides, at most about 120 nucleotides, at most about 150nucleotides, at most about 200 nucleotides, at most about 300nucleotides, at most about 400 nucleotides, at most about 500nucleotides, at most about 600 nucleotides, at most about 700nucleotides, at most about 800 nucleotides, at most about 900nucleotides, or at most about 1000 nucleotides, at most about 1100nucleotides, at most about 1200 nucleotides, at most about 1300nucleotides, at most about 1400 nucleotides, or at most about 1500nucleotides downstream from GRCh37/hg19: chr2:166,863,740. In someembodiments, the ASO targets a sequence more than 300 nucleotidesdownstream from GRCh37/hg19: chr2:166,863,740.

As described herein in the Examples, the SCN1A gene (SEQ ID NO. 1) wasanalyzed for NIE and inclusion of a portion of intron 20 (SEQ ID NO. 4)(this portion is referred as exon 20x throughout the present disclosure)was observed. In some embodiments, the ASOs disclosed herein target aNIE containing pre-mRNA (SEQ ID NO. 2) transcribed from a SCN1A genomicsequence. In some embodiments, the ASO targets a NIE containing pre-mRNAtranscript from a SCN1A genomic sequence comprising a portion of intron20. In some embodiments, the ASO targets a NIE containing pre-mRNAtranscript from a SCN1A genomic sequence comprising exon 20x (SEQ ID NO.6). In some embodiments, the ASO targets a NIE containing pre-mRNAtranscript of SEQ ID NO. 2 or 12. In some embodiments, the ASO targets aNIE containing pre-mRNA transcript of SEQ ID NO. 2 or 12 comprising anNIE. In some embodiments, the ASO targets a NIE containing pre-mRNAtranscript of SEQ ID NO. 2 comprising exon 20x (SEQ ID NO. 10). In someembodiments, the ASOs disclosed herein target a SCN1A pre-mRNA sequence(SEQ ID NO. 2 or 12). In some embodiments, the ASO targets a SCN1Apre-mRNA sequence comprising an NIE (SEQ ID NO. 10 or 20). In someembodiments, the ASO targets a SCN1A pre-mRNA sequence according to anyone of SEQ ID NOs: 7-10 or 17-20. In some embodiments, the ASO has asequence according to any one of SEQ ID NOs: 21-67. In some embodiments,the ASO has a sequence according to any one of SEQ ID NOs: 68-114. Insome embodiments, the ASO has a sequence according to any one of SEQ IDNOs: 115-209. In some embodiments, the ASO has a sequence according toany one of SEQ ID NOs: 210-256. In some embodiments, the ASO has asequence according to any one of SEQ ID NOs: 257-303. In someembodiments, the ASO has a sequence according to any one of SEQ ID NOs:304-341. In some embodiments, the ASO has a sequence according to anyone of SEQ ID NOs: 342-379.

In some embodiments, the SCN1A NIE containing pre-mRNA transcript isencoded by a genetic sequence with at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.: 1 or 11. Insome embodiments, the SCN1A NIE pre-mRNA transcript comprises a sequencewith at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to any one of SEQ ID NOs.: 2-10 and 12-20.

In some embodiments, the SCN1A NIE containing pre-mRNA transcript (orNMD exon mRNA) comprises a sequence with at least about 80%, 85%, 90%,95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 2, 7-10,12, and 17-20. In some embodiments, SCN1A NIE containing pre-mRNAtranscript (or NMD exon mRNA) is encoded by a sequence with at leastabout 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NOs:1, 3-6, 11, and 13-16. In some embodiments, the targeted portion of theNMD exon mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,97%, or 100% sequence identity to a region comprising at least 8contiguous nucleic acids of SEQ ID NOs: 2, 7-10, 12, and 17-20.

In some embodiments, the ASO targets exon 20 of a SCN1A NIE containingpre-mRNA comprising NIE exon 20x. In some embodiments, the ASO targetsan exon 21 sequence downstream (or 3′) of NIE exon 20x. In someembodiments, the ASO targets a sequence about 4 to about 300 nucleotidesupstream (or 5′) from the 5′ end of exon 20x. In some embodiments, theASO targets a sequence about 4 to about 300 nucleotides downstream (or3′) from the 3′ end of exon 20x. In some embodiments, the ASO has asequence according to any one of SEQ ID NOs: 21-67. In some embodiments,the ASO has a sequence according to any one of SEQ ID NOs: 210-256.

In some embodiments, the ASO targets a sequence upstream from the 5′ endof an NIE. For example, ASOs targeting a sequence upstream from the 5′end of an NIE (e.g. exon 20x in human SCN1A, or exon 21x in mouse SCN1A)can comprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100%sequence identity to any one of SEQ ID NOs: 21-38. For another example,ASOs targeting a sequence upstream from the 5′ end of an NIE (e.g. exon20x in human SCN1A, or exon 21x in mouse SCN1A) can comprise a sequencewith at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to anyone of SEQ ID NOs: 68-85. In some embodiments, the ASOs target asequence containing a exon-intron boundary (or junction). For example,ASOs targeting a sequence containing an exon-intron boundary cancomprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100%sequence identity to any one of SEQ ID NOs: 39-41, 51, 52, 228-230, 240,or 241. For another example, ASOs targeting a sequence containing anexon-intron boundary can comprise a sequence with at least 80%, 85%,90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 86-88and 98-99. In some embodiments, the ASOs target a sequence downstreamfrom the 3′ end of an NIE. For example, ASOs targeting a sequencedownstream from the 3′ end of an NIE (e.g. exon 20x in human SCN1A, orexon 21x in mouse SCN1A) can comprise a sequence with at least 80%, 85%,90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs:53-67. For another example, ASOs targeting a sequence downstream fromthe 3′ end of an NIE (e.g. exon 20x in human SCN1A, or exon 21x in mouseSCN1A) can comprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or100% sequence identity to any one of SEQ ID NOs: 100-114. In someembodiments, ASOs target a sequence within an NIE. For example, ASOstargeting a sequence within an NIE (e.g. exon 20x in human SCN1A, orexon 21x in mouse SCN1A) can comprise a sequence with at least 80%, 85%,90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs:42-50, or 231-239. For another example, ASOs targeting a sequence withinan NIE (e.g. exon 20x in human SCN1A, or exon 21x in mouse SCN1A) cancomprise a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100%sequence identity to any one of SEQ ID NOs: 89-97.

In some embodiments, the ASO targets exon 20x in a SCN1A NIE containingpre-mRNA comprising exon 20x. In some embodiments, the ASO targets anexon 20x sequence downstream (or 3′) from the 5′ end of the exon 20x ofa SCN1A pre-mRNA. In some embodiments, the ASO targets an exon 20xsequence upstream (or 5′) from the 3′ end of the exon 20x of a SCN1Apre-mRNA.

In some embodiments, the targeted portion of the SCN1A NIE containingpre-mRNA is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 (intron numberingcorresponding to the mRNA sequence at NM_006920). In some embodiments,hybridization of an ASO to the targeted portion of the NIE pre-mRNAresults in exon skipping of at least one of NIE within intron 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25, and subsequently increases SCN1A protein production. Insome embodiments, hybridization of an ASO to the targeted portion of theNIE pre-mRNA inhibits or blocks exon skipping of at least one of NIEwithin intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25, and subsequently decreases SCN1Aprotein production. In some embodiments, the targeted portion of theSCN1A NIE containing pre-mRNA is in intron 20. One of skill in the artcan determine the corresponding intron number in any isoform based on anintron sequence provided herein or using the number provided inreference to the mRNA sequence at NM_006920, NM_001202435, NM_001165964,or NM_001165963. One of skill in the art also can determine thesequences of flanking exons in any SCN1A isoform for targeting using themethods of the invention, based on an intron sequence provided herein orusing the intron number provided in reference to the mRNA sequence atNM_006920, NM_001202435, NM_001165964, or NM_001165963.

In some embodiments, the methods and compositions of the presentdisclosure are used to modulate, e.g., increase or decrease, theexpression of SCN1A by inducing or inhibiting exon skipping of apseudo-exon of an SCN1A NIE containing pre-mRNA. In some embodiments,the pseudo-exon is a sequence within any of introns 1-25. In someembodiments, the pseudo-exon is a sequence within any of introns 2, 4,6, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, and 25. In someembodiments, the pseudo-exon is a sequence within any of introns 15, 18,and 19. In some embodiments, the pseudo-exon can be any SCN1A intron ora portion thereof. In some embodiments, the pseudo-exon is within intron20. The SCN1A intron numbering used herein corresponds to the mRNAsequence at NM_006920. It is understood that the intron numbering maychange in reference to a different SCN1A isoform sequence.

SCN1A Protein

The SCN1A gene can encode SCN1A (sodium channel, voltage-gated, type I,alpha subunit) protein, which can also be referred to as alpha-subunitof voltage-gated sodium channel Nav1.1. Also described above, SCN1Amutations in DS are spread across the entire protein. More than 100novel mutations have been identified throughout the gene with the moredebilitating arising de novo. These comprise of truncations (47%),missense (43%), deletions (3%), and splice site mutations (7%). Thepercentage of subjects carrying SCN1A mutations varies between 33 and100%. The majority of mutations are novel changes (88%).

In some embodiments, the methods described herein are used to modulate,e.g., increase or decrease, the production of a functional SCN1Aprotein. As used herein, the term “functional” refers to the amount ofactivity or function of a SCN1A protein that is necessary to eliminateany one or more symptoms of a treated condition, e.g., Dravet syndrome;Epilepsy, generalized, with febrile seizures plus, type 2; Febrileseizures, familial, 3A; Autism; Epileptic encephalopathy, earlyinfantile, 13; Sick sinus syndrome 1; Alzheimer's disease; or SUDEP. Insome embodiments, the methods are used to increase the production of apartially functional SCN1A protein. As used herein, the term “partiallyfunctional” refers to any amount of activity or function of the SCN1Aprotein that is less than the amount of activity or function that isnecessary to eliminate or prevent any one or more symptoms of a diseaseor condition. In some embodiments, a partially functional protein or RNAwill have at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% less activity relative to thefully functional protein or RNA.

In some embodiments, the method is a method of increasing the expressionof the SCN1A protein by cells of a subject having a NIE containingpre-mRNA encoding the SCN1A protein, wherein the subject has Dravetsyndrome caused by a deficient amount of activity of SCN1A protein, andwherein the deficient amount of the SCN1A protein is caused byhaploinsufficiency of the SCN1A protein. In such an embodiment, thesubject has a first allele encoding a functional SCN1A protein, and asecond allele from which the SCN1A protein is not produced. In anothersuch embodiment, the subject has a first allele encoding a functionalSCN1A protein, and a second allele encoding a nonfunctional SCN1Aprotein. In another such embodiment, the subject has a first alleleencoding a functional SCN1A protein, and a second allele encoding apartially functional SCN1A protein. In any of these embodiments, theantisense oligomer binds to a targeted portion of the NIE containingpre-mRNA transcribed from the second allele, thereby inducing exonskipping of the pseudo-exon from the pre-mRNA, and causing an increasein the level of mature mRNA encoding functional SCN1A protein, and anincrease in the expression of the SCN1A protein in the cells of thesubject.

In related embodiments, the method is a method of using an ASO toincrease the expression of a protein or functional RNA. In someembodiments, an ASO is used to increase the expression of SCN1A proteinin cells of a subject having a NIE containing pre-mRNA encoding SCN1Aprotein, wherein the subject has a deficiency, e.g., Dravet Syndrome(DS) (also known as SMEI); severe myoclonic epilepsy of infancy(SMEI)-borderland (SMEB); Febrile seizure (FS); epilepsy, generalized,with febrile seizures plus (GEFS+); epileptic encephalopathy, earlyinfantile, 13; cryptogenic generalized epilepsy; cryptogenic focalepilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; Westsyndrome; idiopathic spasms; early myoclonic encephalopathy; progressivemyoclonic epilepsy; alternating hemiplegia of childhood; unclassifiedepileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP);sick sinus syndrome 1; early infantile SCN1A encephalopathy; earlyinfantile epileptic encephalopathy (EIEE); or autism, in the amount orfunction of a SCN1A protein. In some embodiments, an ASO is used toincrease the expression of SCN1A protein in cells of a subject, whereinthe subject has a deficiency, e.g., Epileptic encephalopathy, earlyinfantile, 13; in the amount or function of a SCN8A protein. In someembodiments, an ASO is used to increase the expression of SCN1A proteinin cells of a subject, wherein the subject has a deficiency, e.g., Sicksinus syndrome 1; in the amount or function of a SCN5A protein.

In some embodiments, the NIE containing pre-mRNA transcript that encodesthe protein that is causative of the disease or condition is targeted bythe ASOs described herein. In some embodiments, a NIE containingpre-mRNA transcript that encodes a protein that is not causative of thedisease is targeted by the ASOs. For example, a disease that is theresult of a mutation or deficiency of a first protein in a particularpathway may be ameliorated by targeting a NIE containing pre-mRNA thatencodes a second protein, thereby increasing production of the secondprotein. In some embodiments, the function of the second protein is ableto compensate for the mutation or deficiency of the first protein (whichis causative of the disease or condition).

In some embodiments, the subject has:

(a) a first mutant allele from which

-   -   (i) the SCN1A protein is produced at a reduced level compared to        production from a wild-type allele,    -   (ii) the SCN1A protein is produced in a form having reduced        function compared to an equivalent wild-type protein, or    -   (iii) the SCN1A protein or functional RNA is not produced; and

(b) a second mutant allele from which

-   -   (i) the SCN1A protein is produced at a reduced level compared to        production from a wild-type allele,    -   (ii) the SCN1A protein is produced in a form having reduced        function compared to an equivalent wild-type protein, or    -   (iii) the SCN1A protein is not produced, and        wherein the NIE containing pre-mRNA is transcribed from the        first allele and/or the second allele. In these embodiments, the        ASO binds to a targeted portion of the NIE containing pre-mRNA        transcribed from the first allele or the second allele, thereby        inducing exon skipping of the pseudo-exon from the NIE        containing pre-mRNA, and causing an increase in the level of        mRNA encoding SCN1A protein and an increase in the expression of        the target protein or functional RNA in the cells of the        subject. In these embodiments, the target protein or functional        RNA having an increase in expression level resulting from the        exon skipping of the pseudo-exon from the NIE containing        pre-mRNA is either in a form having reduced function compared to        the equivalent wild-type protein (partially-functional), or        having full function compared to the equivalent wild-type        protein (fully-functional).

In some embodiments, the level of mRNA encoding SCN1A protein isincreased 1.1 to 10-fold, when compared to the amount of mRNA encodingSCN1A protein that is produced in a control cell, e.g., one that is nottreated with the antisense oligomer or one that is treated with anantisense oligomer that does not bind to the targeted portion of theSCN1A NIE containing pre-mRNA.

In some embodiments, a subject treated using the methods of the presentdisclosure expresses a partially functional SCN1A protein from oneallele, wherein the partially functional SCN1A protein is caused by aframeshift mutation, a nonsense mutation, a missense mutation, or apartial gene deletion. In some embodiments, a subject treated using themethods of the invention expresses a nonfunctional SCN1A protein fromone allele, wherein the nonfunctional SCN1A protein is caused by aframeshift mutation, a nonsense mutation, a missense mutation, a partialgene deletion, in one allele. In some embodiments, a subject treatedusing the methods of the invention has a SCN1A whole gene deletion, inone allele.

In some embodiments, the method is a method of decreasing the expressionof the SCN1A protein by cells of a subject having a NIE containingpre-mRNA encoding the SCN1A protein, and wherein the subject has again-of-function mutation in Na_(v)1.1. In such an embodiment, thesubject has an allele from which the SCN1A protein is produced in anelevated amount or an allele encoding a mutant SCN1A that inducesincreased activity of Na_(v)1.1 in the cell. In some embodiments, theincreased activity of Na_(v)1.1 is characterized by a prolonged or nearpersistent sodium current mediated by the mutant Nav1.1 channel, aslowing of fast inactivation, a positive shift in steady-stateinactivation, higher channel availability during repetitive stimulation,increased non-inactivated depolarization-induced persistent sodiumcurrents, delayed entry into inactivation, accelerated recovery fromfast inactivation, and/or rescue of folding defects by incubation atlower temperature or co-expression of interacting proteins. In any ofthese embodiments, the antisense oligomer binds to a targeted portion ofthe NIE containing pre-mRNA transcribed from the second allele, therebyinhibiting or blocking exon skipping of the pseudo-exon from thepre-mRNA, and causing a decrease in the level of mature mRNA encodingfunctional SCN1A protein, and a decrease in the expression of the SCN1Aprotein in the cells of the subject.

In related embodiments, the method is a method of using an ASO todecrease the expression of a protein or functional RNA. In someembodiments, an ASO is used to decrease the expression of SCN1A proteinin cells of a subject having a NIE containing pre-mRNA encoding SCN1Aprotein. In some embodiments, the subject has a gain-of-functionmutation in Nav1.1, e.g., migraine. In some embodiments, an ASO is usedto decrease the expression of SCN1A protein in cells of a subject, thesubject has a gain-of-function mutation in Nav1.1, e.g., migraine,familial hemiplegic, 3.

In some embodiments, the level of mRNA encoding SCN1A protein isdecreased 1.1 to 10-fold, when compared to the amount of mRNA encodingSCN1A protein that is produced in a control cell, e.g., one that is nottreated with the antisense oligomer or one that is treated with anantisense oligomer that does not bind to the targeted portion of theSCN1A NIE containing pre-mRNA.

In some embodiments, a subject treated using the methods of the presentdisclosure expresses a mutant SCN1A protein from one allele, wherein themutant SCN1A protein is caused by a frameshift mutation, a nonsensemutation, a missense mutation, or a partial gene deletion, and whereinthe mutant SCN1A protein causes an elevated activity level of Na_(v)1.1.In some embodiments, a subject treated using the methods of the presentdisclosure expresses an elevated amount of SCN1A protein from one alleledue to a frameshift mutation, a nonsense mutation, a missense mutation,or a partial gene deletion.

In embodiments of the present invention, a subject can have a mutationin SCN1A. Mutations in SCN1A can be spread throughout said gene. SCN1Aprotein can consist of four domains. Said SCN1A domains can havetransmembrane segments. Mutations in said SCN1A protein may arisethroughout said protein. Said SCN1A protein may consist of at least twoisoforms. Mutations in SCN1A may comprise of R931C, R946C, M934I,R1648C, or R1648H. In some cases, mutations may be observed in aC-terminus of a SCN1A protein. Mutations in a SCN1A protein may also befound in loops between segments 5 and 6 of the first three domains ofsaid SCN1A protein. In some cases, mutations may be observed in anN-terminus of a SCN1A protein. Exemplary mutations within SCN1A include,but are not limited to, R222X, R712X, I227S, R1892X, W952X, R1245X,R1407X, W1434R, c.4338+1G>A, S1516X, L1670fsX1678, or K1846fsX1856.Mutations that can be targeted with the present invention may alsoencode a pore of an ion channel.

In some embodiments, the methods and compositions described herein canbe used to treat DS. In other embodiments, the methods and compositionsdescribed herein can be used to treat severe myclonic epilepsy ofinfancy (SMEI). In other embodiments, the methods and compositionsdescribed herein can be used to treat borderline Dravet syndrome;Epilepsy, generalized, with febrile seizures plus, type 2; Febrileseizures, familial, 3A; Migraine, familial hemiplegic, 3; Autism;Epileptic encephalopathy, early infantile, 13; Sick sinus syndrome 1;Alzheimer's disease or SUDEP. The methods and compositions describedherein can also be used to treat borderline SMEI. Additionally, themethods and compositions described herein can be used to treatgeneralized epilepsy with febrile seizures plus (GEFS+). GEFS+ may beassociated with mutations in epilepsy-associated ion channel subunitssuch as SCN1B or GABRG2. The methods and compositions described hereincan also be used to treat sodium channelopathies. Sodium channelopathiesmay be associated with mutations in SCN1A. Sodium channelopathies mayalso be associated with subunits of SCN1A, such as the beta subunit,SCN1B. In some cases, additional diseases associated with SCN1Amutations may also be treated with the present disclosure. Related SCN1Adiseases associated with SCN1A mutations include, but are not limitedto, atypical myotonia congenita, hyperkalemic periodic paralysis, andparamyotonia congenita.

In some embodiments, a subject having any SCN1A mutation known in theart and described in the literature referenced above (e.g., by Hamdan,et al., 2009, Mulley, et al., 2005) can be treated using the methods andcompositions described herein. In some embodiments, the mutation iswithin any SCN1A intron or exon.

Exon Inclusion

As used herein, a “NIE containing pre-mRNA” is a pre-mRNA transcriptthat contains at least one pseudo-exon. Alternative or aberrant splicingcan result in inclusion of the at least one pseudo-exon in the maturemRNA transcripts. The terms “mature mRNA,” and “fully-spliced mRNA,” areused interchangeably herein to describe a fully processed mRNA.Inclusion of the at least one pseudo-exon can be non-productive mRNA andlead to NMD of the mature mRNA. NIE containing mature mRNA may sometimeslead to aberrant protein expression.

In some embodiments, the included pseudo-exon is the most abundantpseudo-exon in a population of NIE containing pre-mRNAs transcribed fromthe gene encoding the target protein in a cell. In some embodiments, theincluded pseudo-exon is the most abundant pseudo-exon in a population ofNIE containing pre-mRNAs transcribed from the gene encoding the targetprotein in a cell, wherein the population of NIE containing pre-mRNAscomprises two or more included pseudo-exons. In some embodiments, anantisense oligomer targeted to the most abundant pseudo-exon in thepopulation of NIE containing pre-mRNAs encoding the target proteininduces exon skipping of one or two or more pseudo-exons in thepopulation, including the pseudo-exon to which the antisense oligomer istargeted or binds. In embodiments, the targeted region is in apseudo-exon that is the most abundant pseudo-exon in a NIE containingpre-mRNA encoding the SCN1A protein.

The degree of exon inclusion can be expressed as percent exon inclusion,e.g., the percentage of transcripts in which a given pseudo-exon isincluded. In brief, percent exon inclusion can be calculated as thepercentage of the amount of RNA transcripts with the exon inclusion,over the sum of the average of the amount of RNA transcripts with exoninclusion plus the average of the amount of RNA transcripts with exonexclusion.

In some embodiments, an included pseudo-exon is an exon that isidentified as an included pseudo-exon based on a determination of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, or at least about 50%, inclusion.In embodiments, a included pseudo-exon is an exon that is identified asa included pseudo-exon based on a determination of about 5% to about100%, about 5% to about 95%, about 5% to about 90%, about 5% to about85%, about 5% to about 80%, about 5% to about 75%, about 5% to about70%, about 5% to about 65%, about 5% to about 60%, about 5% to about55%, about 5% to about 50%, about 5% to about 45%, about 5% to about40%, about 5% to about 35%, about 5% to about 30%, about 5% to about25%, about 5% to about 20%, about 5% to about 15%, about 10% to about100%, about 10% to about 95%, about 10% to about 90%, about 10% to about85%, about 10% to about 80%, about 10% to about 75%, about 10% to about70%, about 10% to about 65%, about 10% to about 60%, about 10% to about55%, about 10% to about 50%, about 10% to about 45%, about 10% to about40%, about 10% to about 35%, about 10% to about 30%, about 10% to about25%, about 10% to about 20%, about 15% to about 100%, about 15% to about95%, about 15% to about 90%, about 15% to about 85%, about 15% to about80%, about 15% to about 75%, about 15% to about 70%, about 15% to about65%, about 15% to about 60%, about 15% to about 55%, about 15% to about50%, about 15% to about 45%, about 15% to about 40%, about 15% to about35%, about 15% to about 30%, about 15% to about 25%, about 20% to about100%, about 20% to about 95%, about 20% to about 90%, about 20% to about85%, about 20% to about 80%, about 20% to about 75%, about 20% to about70%, about 20% to about 65%, about 20% to about 60%, about 20% to about55%, about 20% to about 50%, about 20% to about 45%, about 20% to about40%, about 20% to about 35%, about 20% to about 30%, about 25% to about100%, about 25% to about 95%, about 25% to about 90%, about 25% to about85%, about 25% to about 80%, about 25% to about 75%, about 25% to about70%, about 25% to about 65%, about 25% to about 60%, about 25% to about55%, about 25% to about 50%, about 25% to about 45%, about 25% to about40%, or about 25% to about 35%, inclusion. ENCODE data (described by,e.g., Tilgner, et al., 2012, “Deep sequencing of subcellular RNAfractions shows splicing to be predominantly co-transcriptional in thehuman genome but inefficient for lncRNAs,” Genome Research22(9):1616-25) can be used to aid in identifying exon inclusion.

In some embodiments, contacting cells with an ASO that is complementaryto a targeted portion of a SCN1A pre-mRNA transcript results in anincrease in the amount of SCN1A protein produced by at least 10, 20, 30,40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%,compared to the amount of the protein produced by a cell in the absenceof the ASO/absence of treatment. In some embodiments, the total amountof SCN1A protein produced by the cell to which the antisense oligomer iscontacted is increased about 1.1 to about 10-fold, about 1.5 to about10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 toabout 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold,about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 toabout 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold,about 4 to about 9-fold, at least about 1.1-fold, at least about1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about3-fold, at least about 3.5-fold, at least about 4-fold, at least about5-fold, or at least about 10-fold, compared to the amount of targetprotein produced by a control compound. A control compound can be, forexample, an oligonucleotide that is not complementary to a targetedportion of the pre-mRNA.

In some embodiments, contacting cells with an ASO that is complementaryto a targeted portion of a SCN1A pre-mRNA transcript results in adecrease in the amount of SCN1A protein produced by at least 10, 20, 30,40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%,compared to the amount of the protein produced by a cell in the absenceof the ASO/absence of treatment. In some embodiments, the total amountof SCN1A protein produced by the cell to which the antisense oligomer iscontacted is decreased about 1.1 to about 10-fold, about 1.5 to about10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 toabout 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold,about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 toabout 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold,about 4 to about 9-fold, at least about 1.1-fold, at least about1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about3-fold, at least about 3.5-fold, at least about 4-fold, at least about5-fold, or at least about 10-fold, compared to the amount of targetprotein produced by a control compound. A control compound can be, forexample, an oligonucleotide that is not complementary to a targetedportion of the pre-mRNA.

In some embodiments, contacting cells with an ASO that is complementaryto a targeted portion of a SCN1A pre-mRNA transcript results in anincrease in the amount of mRNA encoding SCN1A, including the mature mRNAencoding the target protein. In some embodiments, the amount of mRNAencoding SCN1A protein, or the mature mRNA encoding the SCN1A protein,is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 500, or 1000%, compared to the amount of the proteinproduced by a cell in the absence of the ASO/absence of treatment. Insome embodiments, the total amount of the mRNA encoding SCN1A protein,or the mature mRNA encoding SCN1A protein produced in the cell to whichthe antisense oligomer is contacted is increased about 1.1 to about10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 toabout 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold,about 4 to about 8-fold, about 4 to about 9-fold, at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about4-fold, at least about 5-fold, or at least about 10-fold compared to theamount of mature RNA produced in an untreated cell, e.g., an untreatedcell or a cell treated with a control compound. A control compound canbe, for example, an oligonucleotide that is not complementary to atargeted portion of the SCN1A NIE containing pre-mRNA.

In some embodiments, contacting cells with an ASO that is complementaryto a targeted portion of a SCN1A pre-mRNA transcript results in adecrease in the amount of mRNA encoding SCN1A, including the mature mRNAencoding the target protein. In some embodiments, the amount of mRNAencoding SCN1A protein, or the mature mRNA encoding the SCN1A protein,is decreased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 500, or 1000%, compared to the amount of the proteinproduced by a cell in the absence of the ASO/absence of treatment. Insome embodiments, the total amount of the mRNA encoding SCN1A protein,or the mature mRNA encoding SCN1A protein produced in the cell to whichthe antisense oligomer is contacted is decreased about 1.1 to about10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 toabout 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold,about 4 to about 8-fold, about 4 to about 9-fold, at least about1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about4-fold, at least about 5-fold, or at least about 10-fold compared to theamount of mature RNA produced in an untreated cell, e.g., an untreatedcell or a cell treated with a control compound. A control compound canbe, for example, an oligonucleotide that is not complementary to atargeted portion of the SCN1A NIE containing pre-mRNA.

The NIE can be in any length. In some embodiments, the NIE comprises afull sequence of an intron, in which case, it can be referred to asintron retention. In some embodiments, the NIE can be a portion of theintron. In some embodiments, the NIE can be a 5′ end portion of anintron including a 5′ss sequence. In some embodiments, the NIE can be a3′ end portion of an intron including a 3′ss sequence. In someembodiments, the NIE can be a portion within an intron without inclusionof a 5′ss sequence. In some embodiments, the NIE can be a portion withinan intron without inclusion of a 3′ss sequence. In some embodiments, theNIE can be a portion within an intron without inclusion of either a 5′ssor a 3′ss sequence. In some embodiments, the NIE can be from 5nucleotides to 10 nucleotides in length, from 10 nucleotides to 15nucleotides in length, from 15 nucleotides to 20 nucleotides in length,from 20 nucleotides to 25 nucleotides in length, from 25 nucleotides to30 nucleotides in length, from 30 nucleotides to 35 nucleotides inlength, from 35 nucleotides to 40 nucleotides in length, from 40nucleotides to 45 nucleotides in length, from 45 nucleotides to 50nucleotides in length, from 50 nucleotides to 55 nucleotides in length,from 55 nucleotides to 60 nucleotides in length, from 60 nucleotides to65 nucleotides in length, from 65 nucleotides to 70 nucleotides inlength, from 70 nucleotides to 75 nucleotides in length, from 75nucleotides to 80 nucleotides in length, from 80 nucleotides to 85nucleotides in length, from 85 nucleotides to 90 nucleotides in length,from 90 nucleotides to 95 nucleotides in length, or from 95 nucleotidesto 100 nucleotides in length. In some embodiments, the NIE can be atleast 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides,at least 40 nucleotides, at least 50 nucleotides, at least 60nucleotides, at least 70 nucleotides, at least 80 nucleotides in length,at least 90 nucleotides, or at least 100 nucleotides in length. In someembodiments, the NIE can be from 100 to 200 nucleotides in length, from200 to 300 nucleotides in length, from 300 to 400 nucleotides in length,from 400 to 500 nucleotides in length, from 500 to 600 nucleotides inlength, from 600 to 700 nucleotides in length, from 700 to 800nucleotides in length, from 800 to 900 nucleotides in length, from 900to 1,000 nucleotides in length. In some embodiments, the NIE may belonger than 1,000 nucleotides in length.

Inclusion of a pseudo-exon can lead to a frameshift and the introductionof a premature termination codon (PIC) in the mature mRNA transcriptrendering the transcript a target of NMD. Mature mRNA transcriptcontaining NIE can be non-productive mRNA transcript which does not leadto protein expression. The PIC can be present in any position downstreamof an NIE. In some embodiments, the PIC can be present in any exondownstream of an NIE. In some embodiments, the PIC can be present withinthe NIE. For example, inclusion of exon 20x in an mRNA transcriptencoded by the SCN1A gene can induce a PIC in the mRNA transcript, e.g.,a PIC in exon 21 of the mRNA transcript.

Therapeutic Agents

In various embodiments of the present disclosure, compositions andmethods comprising a therapeutic agent are provided to modulate proteinexpression level of SCN1A. In some embodiments, provided herein arecompositions and methods to modulate alternative splicing of SCN1Apre-mRNA. In some embodiments, provided herein are compositions andmethods to induce exon skipping in the splicing of SCN1A pre-mRNA, e.g.,to induce skipping of a pseudo-exon during splicing of SCN1A pre-mRNA.In other embodiments, therapeutic agents may be used to induce theinclusion of an exon in order to decrease the protein expression level.

In some embodiment, a therapeutic agent disclosed herein is a smallmolecule, a polypeptide, or a polynucleic acid polymer. In someinstances, the therapeutic agent is a small molecule. In some instances,the therapeutic agent is a polypeptide. In some instances, thetherapeutic agent is a polynucleic acid polymer. In some cases, thetherapeutic agent is a repressor agent. In additional cases, thetherapeutic agent is an enhancer agent.

A therapeutic agent disclosed herein can be a NIE repressor agent. Atherapeutic agent may comprise a polynucleic acid polymer.

According to one aspect of the present disclosure, provided herein is amethod of treatment or prevention of a condition associated with afunctional-SCN1A protein deficiency, comprising administering a NIErepressor agent to a subject to increase levels of functional SCN1Aprotein, wherein the agent binds to a region of the pre-mRNA transcriptto decrease inclusion of the NIE in the mature transcript. For example,provided herein is a method of treatment or prevention of a conditionassociated with a functional-SCN1A protein deficiency, comprisingadministering a NIE repressor agent to a subject to increase levels offunctional SCN1A protein, wherein the agent binds to a region of anintron containing an NIE (e.g., intron 20 in human SCN1A gene) of thepre-mRNA transcript or to a NIE-activating regulatory sequence in thesame intron.

Where reference is made to reducing NIE inclusion in the mature mRNA,the reduction may be complete, e.g., 100%, or may be partial. Thereduction may be clinically significant. The reduction/correction may berelative to the level of NIE inclusion in the subject without treatment,or relative to the amount of NIE inclusion in a population of similarsubjects. The reduction/correction may be at least 10% less NIEinclusion relative to the average subject, or the subject prior totreatment. The reduction may be at least 20% less NIE inclusion relativeto an average subject, or the subject prior to treatment. The reductionmay be at least 40% less NIE inclusion relative to an average subject,or the subject prior to treatment. The reduction may be at least 50%less NIE inclusion relative to an average subject, or the subject priorto treatment. The reduction may be at least 60% less NIE inclusionrelative to an average subject, or the subject prior to treatment. Thereduction may be at least 80% less NIE inclusion relative to an averagesubject, or the subject prior to treatment. The reduction may be atleast 90% less NIE inclusion relative to an average subject, or thesubject prior to treatment.

Where reference is made to increasing active-SCN1A protein levels, theincrease may be clinically significant. The increase may be relative tothe level of active-SCN1A protein in the subject without treatment, orrelative to the amount of active-SCN1A protein in a population ofsimilar subjects. The increase may be at least 10% more active-SCN1Aprotein relative to the average subject, or the subject prior totreatment. The increase may be at least 20% more active-SCN1A proteinrelative to the average subject, or the subject prior to treatment. Theincrease may be at least 40% more active-SCN1A protein relative to theaverage subject, or the subject prior to treatment. The increase may beat least 50% more active-SCN1A protein relative to the average subject,or the subject prior to treatment. The increase may be at least 80% moreactive-SCN1A protein relative to the average subject, or the subjectprior to treatment. The increase may be at least 100% more active-SCN1Aprotein relative to the average subject, or the subject prior totreatment. The increase may be at least 200% more active-SCN1A proteinrelative to the average subject, or the subject prior to treatment. Theincrease may be at least 500% more active-SCN1A protein relative to theaverage subject, or the subject prior to treatment.

In embodiments wherein the NIE repressor agent comprises a polynucleicacid polymer, the polynucleic acid polymer may be about 50 nucleotidesin length. The polynucleic acid polymer may be about 45 nucleotides inlength. The polynucleic acid polymer may be about 40 nucleotides inlength. The polynucleic acid polymer may be about 35 nucleotides inlength. The polynucleic acid polymer may be about 30 nucleotides inlength. The polynucleic acid polymer may be about 24 nucleotides inlength. The polynucleic acid polymer may be about 25 nucleotides inlength. The polynucleic acid polymer may be about 20 nucleotides inlength. The polynucleic acid polymer may be about 19 nucleotides inlength. The polynucleic acid polymer may be about 18 nucleotides inlength. The polynucleic acid polymer may be about 17 nucleotides inlength. The polynucleic acid polymer may be about 16 nucleotides inlength. The polynucleic acid polymer may be about 15 nucleotides inlength. The polynucleic acid polymer may be about 14 nucleotides inlength. The polynucleic acid polymer may be about 13 nucleotides inlength. The polynucleic acid polymer may be about 12 nucleotides inlength. The polynucleic acid polymer may be about 11 nucleotides inlength. The polynucleic acid polymer may be about 10 nucleotides inlength. The polynucleic acid polymer may be between about 10 and about50 nucleotides in length. The polynucleic acid polymer may be betweenabout 10 and about 45 nucleotides in length. The polynucleic acidpolymer may be between about 10 and about 40 nucleotides in length. Thepolynucleic acid polymer may be between about 10 and about 35nucleotides in length. The polynucleic acid polymer may be between about10 and about 30 nucleotides in length. The polynucleic acid polymer maybe between about 10 and about 25 nucleotides in length. The polynucleicacid polymer may be between about 10 and about 20 nucleotides in length.The polynucleic acid polymer may be between about 15 and about 25nucleotides in length. The polynucleic acid polymer may be between about15 and about 30 nucleotides in length. The polynucleic acid polymer maybe between about 12 and about 30 nucleotides in length.

The sequence of the polynucleic acid polymer may be at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 99.5% complementary to a target sequence of an mRNAtranscript, e.g., a partially processed mRNA transcript. The sequence ofthe polynucleic acid polymer may be 100% complementary to a targetsequence of a pre-mRNA transcript.

The sequence of the polynucleic acid polymer may have 4 or fewermismatches to a target sequence of the pre-mRNA transcript. The sequenceof the polynucleic acid polymer may have 3 or fewer mismatches to atarget sequence of the pre-mRNA transcript. The sequence of thepolynucleic acid polymer may have 2 or fewer mismatches to a targetsequence of the pre-mRNA transcript. The sequence of the polynucleicacid polymer may have 1 or fewer mismatches to a target sequence of thepre-mRNA transcript. The sequence of the polynucleic acid polymer mayhave no mismatches to a target sequence of the pre-mRNA transcript.

The polynucleic acid polymer may specifically hybridize to a targetsequence of the pre-mRNA transcript. For example, the polynucleic acidpolymer may have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or100% sequence complementarity to a target sequence of the pre-mRNAtranscript. The hybridization may be under high stringent hybridizationconditions.

The polynucleic acid polymer may have a sequence with at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 99.5% sequence identity to a sequence selected from thegroup consisting of SEQ ID NOs: 21-67. The polynucleic acid polymer mayhave a sequence with 100% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 21-67. In some instances, thepolynucleic acid polymer may have a sequence with at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 99.5% sequence identity to a sequence selected from thegroup consisting of SEQ ID NOs: 68-114. In some cases, the polynucleicacid polymer may have a sequence with 100% sequence identity to asequence selected from the group consisting of SEQ ID NOs: 68-114.

Where reference is made to a polynucleic acid polymer sequence, theskilled person will understand that one or more substitutions may betolerated, optionally two substitutions may be tolerated in thesequence, such that it maintains the ability to hybridize to the targetsequence; or where the substitution is in a target sequence, the abilityto be recognized as the target sequence. References to sequence identitymay be determined by BLAST sequence alignment using standard/defaultparameters. For example, the sequence may have 99% identity and stillfunction according to the present disclosure. In other embodiments, thesequence may have 98% identity and still function according to thepresent disclosure. In another embodiment, the sequence may have 95%identity and still function according to the present disclosure. Inanother embodiment, the sequence may have 90% identity and stillfunction according to the present disclosure.

Antisense Oligomers

Provided herein is a composition comprising an antisense oligomer thatinduces exon skipping by binding to a targeted portion of a SCN1A NIEcontaining pre-mRNA. As used herein, the terms “ASO” and “antisenseoligomer” are used interchangeably and refer to an oligomer such as apolynucleotide, comprising nucleobases that hybridizes to a targetnucleic acid (e.g., a SCN1A NIE containing pre-mRNA) sequence byWatson-Crick base pairing or wobble base pairing (G-U). The ASO may haveexact sequence complementary to the target sequence or nearcomplementarity (e.g., sufficient complementarity to bind the targetsequence and enhancing splicing at a splice site). ASOs are designed sothat they bind (hybridize) to a target nucleic acid (e.g., a targetedportion of a pre-mRNA transcript) and remain hybridized underphysiological conditions. Typically, if they hybridize to a site otherthan the intended (targeted) nucleic acid sequence, they hybridize to alimited number of sequences that are not a target nucleic acid (to a fewsites other than a target nucleic acid). Design of an ASO can take intoconsideration the occurrence of the nucleic acid sequence of thetargeted portion of the pre-mRNA transcript or a sufficiently similarnucleic acid sequence in other locations in the genome or cellularpre-mRNA or transcriptome, such that the likelihood the ASO will bindother sites and cause “off-target” effects is limited. Any antisenseoligomers known in the art, for example in PCT Application No.PCT/US2014/054151, published as WO 2015/035091, titled “ReducingNonsense-Mediated mRNA Decay,” incorporated by reference herein, can beused to practice the methods described herein.

In some embodiments, ASOs “specifically hybridize” to or are “specific”to a target nucleic acid or a targeted portion of a NIE containingpre-mRNA. Typically such hybridization occurs with a T_(m) substantiallygreater than 37° C., preferably at least 50° C., and typically between60° C. to approximately 90° C. Such hybridization preferably correspondsto stringent hybridization conditions. At a given ionic strength and pH,the T_(m) is the temperature at which 50% of a target sequencehybridizes to a complementary oligonucleotide.

Oligomers, such as oligonucleotides, are “complementary” to one anotherwhen hybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. A double-stranded polynucleotide can be“complementary” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.Complementarity (the degree to which one polynucleotide is complementarywith another) is quantifiable in terms of the proportion (e.g., thepercentage) of bases in opposing strands that are expected to formhydrogen bonds with each other, according to generally acceptedbase-pairing rules. The sequence of an antisense oligomer (ASO) need notbe 100% complementary to that of its target nucleic acid to hybridize.In certain embodiments, ASOs can comprise at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an ASO in which 18 of 20 nucleobases of theoligomeric compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining non-complementarynucleobases may be clustered together or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. Percent complementarity of an ASO with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul, et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

An ASO need not hybridize to all nucleobases in a target sequence andthe nucleobases to which it does hybridize may be contiguous ornoncontiguous. ASOs may hybridize over one or more segments of apre-mRNA transcript, such that intervening or adjacent segments are notinvolved in the hybridization event (e.g., a loop structure or hairpinstructure may be formed). In certain embodiments, an ASO hybridizes tononcontiguous nucleobases in a target pre-mRNA transcript. For example,an ASO can hybridize to nucleobases in a pre-mRNA transcript that areseparated by one or more nucleobase(s) to which the ASO does nothybridize.

The ASOs described herein comprise nucleobases that are complementary tonucleobases present in a targeted portion of a NIE containing pre-mRNA.The term ASO embodies oligonucleotides and any other oligomeric moleculethat comprises nucleobases capable of hybridizing to a complementarynucleobase on a target mRNA but does not comprise a sugar moiety, suchas a peptide nucleic acid (PNA). The ASOs may comprisenaturally-occurring nucleotides, nucleotide analogs, modifiednucleotides, or any combination of two or three of the preceding. Theterm “naturally occurring nucleotides” includes deoxyribonucleotides andribonucleotides. The term “modified nucleotides” includes nucleotideswith modified or substituted sugar groups and/or having a modifiedbackbone. In some embodiments, all of the nucleotides of the ASO aremodified nucleotides. Chemical modifications of ASOs or components ofASOs that are compatible with the methods and compositions describedherein will be evident to one of skill in the art and can be found, forexample, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No. 5,656,612, U.S.Patent Publication No. 2012/0190728, and Dias and Stein, Mol. CancerTher. 2002, 347-355, herein incorporated by reference in their entirety.

One or more nucleobases of an ASO may be any naturally occurring,unmodified nucleobase such as adenine, guanine, cytosine, thymine anduracil, or any synthetic or modified nucleobase that is sufficientlysimilar to an unmodified nucleobase such that it is capable of hydrogenbonding with a nucleobase present on a target pre-mRNA. Examples ofmodified nucleobases include, without limitation, hypoxanthine,xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5-methylcytosine, and5-hydroxymethoylcytosine.

The ASOs described herein also comprise a backbone structure thatconnects the components of an oligomer. The term “backbone structure”and “oligomer linkages” may be used interchangeably and refer to theconnection between monomers of the ASO. In naturally occurringoligonucleotides, the backbone comprises a 3′-5′ phosphodiester linkageconnecting sugar moieties of the oligomer. The backbone structure oroligomer linkages of the ASOs described herein may include (but are notlimited to) phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,phosphoramidate, and the like. See, e.g., LaPlanche, et al., NucleicAcids Res. 14:9081 (1986); Stec, et al., J. Am. Chem. Soc. 106:6077(1984), Stein, et al., Nucleic Acids Res. 16:3209 (1988), Zon, et al.,Anti-Cancer Drug Design 6:539 (1991); Zon, et al., Oligonucleotides andAnalogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., OxfordUniversity Press, Oxford England (1991)); Stec, et al., U.S. Pat. No.5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In someembodiments, the backbone structure of the ASO does not containphosphorous but rather contains peptide bonds, for example in a peptidenucleic acid (PNA), or linking groups including carbamate, amides, andlinear and cyclic hydrocarbon groups. In some embodiments, the backbonemodification is a phosphothioate linkage. In some embodiments, thebackbone modification is a phosphoramidate linkage.

In embodiments, the stereochemistry at each of the phosphorusinternucleotide linkages of the ASO backbone is random. In embodiments,the stereochemistry at each of the phosphorus internucleotide linkagesof the ASO backbone is controlled and is not random. For example, U.S.Pat. App. Pub. No. 2014/0194610, “Methods for the Synthesis ofFunctionalized Nucleic Acids,” incorporated herein by reference,describes methods for independently selecting the handedness ofchirality at each phosphorous atom in a nucleic acid oligomer. Inembodiments, an ASO used in the methods of the invention, including, butnot limited to, any of the ASOs set forth herein in Tables 5 and 6,comprises an ASO having phosphorus internucleotide linkages that are notrandom. In embodiments, a composition used in the methods of theinvention comprises a pure diastereomeric ASO. In embodiments, acomposition used in the methods of the invention comprises an ASO thathas diastereomeric purity of at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99%, about 100%, about 90% to about 100%, about 91% to about100%, about 92% to about 100%, about 93% to about 100%, about 94% toabout 100%, about 95% to about 100%, about 96% to about 100%, about 97%to about 100%, about 98% to about 100%, or about 99% to about 100%.

In embodiments, the ASO has a nonrandom mixture of Rp and Spconfigurations at its phosphorus internucleotide linkages. For example,it has been suggested that a mix of Rp and Sp is required in antisenseoligonucleotides to achieve a balance between good activity and nucleasestability (Wan, et al., 2014, “Synthesis, biophysical properties andbiological activity of second generation antisense oligonucleotidescontaining chiral phosphorothioate linkages,” Nucleic Acids Research42(22): 13456-13468, incorporated herein by reference). In embodiments,an ASO used in the methods of the invention, including, but not limitedto, any of the ASOs set forth herein in SEQ ID NOs: 21-114, comprisesabout 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at leastabout 15% Rp, at least about 20% Rp, at least about 25% Rp, at leastabout 30% Rp, at least about 35% Rp, at least about 40% Rp, at leastabout 45% Rp, at least about 50% Rp, at least about 55% Rp, at leastabout 60% Rp, at least about 65% Rp, at least about 70% Rp, at leastabout 75% Rp, at least about 80% Rp, at least about 85% Rp, at leastabout 90% Rp, or at least about 95% Rp, with the remainder Sp, or about100% Rp. In embodiments, an ASO used in the methods of the invention,including, but not limited to, any of the ASOs set forth herein in SEQID NOs: 21-114, comprises about 10% to about 100% Rp, about 15% to about100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60%Rp, or about 45% to about 55% Rp, with the remainder Sp.

In embodiments, an ASO used in the methods of the invention, including,but not limited to, any of the ASOs set forth herein in SEQ ID NOs:21-114, comprises about 5-100% Sp, at least about 5% Sp, at least about10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25%Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp,at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, atleast about 60% Sp, at least about 65% Sp, at least about 70% Sp, atleast about 75% Sp, at least about 80% Sp, at least about 85% Sp, atleast about 90% Sp, or at least about 95% Sp, with the remainder Rp, orabout 100% Sp. In embodiments, an ASO used in the methods of theinvention, including, but not limited to, any of the ASOs set forthherein in SEQ ID NOs: 21-114, comprises about 10% to about 100% Sp,about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% toabout 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp,about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% toabout 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp,about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% toabout 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp,about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% toabout 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp,about 40% to about 60% Sp, or about 45% to about 55% Sp, with theremainder Rp.

Any of the ASOs described herein may contain a sugar moiety thatcomprises ribose or deoxyribose, as present in naturally occurringnucleotides, or a modified sugar moiety or sugar analog, including amorpholine ring. Non-limiting examples of modified sugar moietiesinclude 2′ substitutions such as 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′MOE), 2′-O-aminoethyl, 2′F; N3′->P5′phosphoramidate, 2′dimethylaminooxyethoxy, 2′dimethylaminoethoxyethoxy,2′-guanidinidium, 2′-O-guanidinium ethyl, carbamate modified sugars, andbicyclic modified sugars. In some embodiments, the sugar moietymodification is selected from 2′-O-Me, 2′F, and 2′MOE. In someembodiments, the sugar moiety modification is an extra bridge bond, suchas in a locked nucleic acid (LNA). In some embodiments the sugar analogcontains a morpholine ring, such as phosphorodiamidate morpholino (PMO).In some embodiments, the sugar moiety comprises a ribofuransyl or2′deoxyribofuransyl modification. In some embodiments, the sugar moietycomprises 2′4′-constrained 2′O-methyloxyethyl (cMOE) modifications. Insome embodiments, the sugar moiety comprises cEt 2′, 4′ constrained 2′-Oethyl BNA modifications. In some embodiments, the sugar moiety comprisestricycloDNA (tcDNA) modifications. In some embodiments, the sugar moietycomprises ethylene nucleic acid (ENA) modifications. In someembodiments, the sugar moiety comprises MCE modifications. Modificationsare known in the art and described in the literature, e.g., by Jarver,et al., 2014, “A Chemical View of Oligonucleotides for Exon Skipping andRelated Drug Applications,” Nucleic Acid Therapeutics 24(1): 37-47,incorporated by reference for this purpose herein.

In some embodiments, each monomer of the ASO is modified in the sameway, for example each linkage of the backbone of the ASO comprises aphosphorothioate linkage or each ribose sugar moiety comprises a2′O-methyl modification. Such modifications that are present on each ofthe monomer components of an ASO are referred to as “uniformmodifications.” In some examples, a combination of differentmodifications may be desired, for example, an ASO may comprise acombination of phosphorodiamidate linkages and sugar moieties comprisingmorpholine rings (morpholinos). Combinations of different modificationsto an ASO are referred to as “mixed modifications” or “mixedchemistries.”

In some embodiments, the ASO comprises one or more backbonemodifications. In some embodiments, the ASO comprises one or more sugarmoiety modification. In some embodiments, the ASO comprises one or morebackbone modifications and one or more sugar moiety modifications. Insome embodiments, the ASO comprises a 2′MOE modification and aphosphorothioate backbone. In some embodiments, the ASO comprises aphosphorodiamidate morpholino (PMO). In some embodiments, the ASOcomprises a peptide nucleic acid (PNA). Any of the ASOs or any componentof an ASO (e.g., a nucleobase, sugar moiety, backbone) described hereinmay be modified in order to achieve desired properties or activities ofthe ASO or reduce undesired properties or activities of the ASO. Forexample, an ASO or one or more components of any ASO may be modified toenhance binding affinity to a target sequence on a pre-mRNA transcript;reduce binding to any non-target sequence; reduce degradation bycellular nucleases (i.e., RNase H); improve uptake of the ASO into acell and/or into the nucleus of a cell; alter the pharmacokinetics orpharmacodynamics of the ASO; and/or modulate the half-life of the ASO.

In some embodiments, the ASOs are comprised of 2′-O-(2-methoxyethyl)(MOE) phosphorothioate-modified nucleotides. ASOs comprised of suchnucleotides are especially well-suited to the methods disclosed herein;oligomers having such modifications have been shown to havesignificantly enhanced resistance to nuclease degradation and increasedbioavailability, making them suitable, for example, for oral delivery insome embodiments described herein. See e.g., Geary, et al., J PharmacolExp Ther. 2001; 296(3):890-7; Geary, et al., J Pharmacol Exp Ther. 2001;296(3):898-904.

Methods of synthesizing ASOs will be known to one of skill in the art.Alternatively or in addition, ASOs may be obtained from a commercialsource.

Unless specified otherwise, the left-hand end of single-stranded nucleicacid (e.g., pre-mRNA transcript, oligonucleotide, ASO, etc.) sequencesis the 5′ end and the left-hand direction of single or double-strandednucleic acid sequences is referred to as the 5′ direction. Similarly,the right-hand end or direction of a nucleic acid sequence (single ordouble stranded) is the 3′ end or direction. Generally, a region orsequence that is 5′ to a reference point in a nucleic acid is referredto as “upstream,” and a region or sequence that is 3′ to a referencepoint in a nucleic acid is referred to as “downstream.” Generally, the5′ direction or end of an mRNA is where the initiation or start codon islocated, while the 3′ end or direction is where the termination codon islocated. In some aspects, nucleotides that are upstream of a referencepoint in a nucleic acid may be designated by a negative number, whilenucleotides that are downstream of a reference point may be designatedby a positive number. For example, a reference point (e.g., an exon-exonjunction in mRNA) may be designated as the “zero” site, and a nucleotidethat is directly adjacent and upstream of the reference point isdesignated “minus one,” e.g., “4,” while a nucleotide that is directlyadjacent and downstream of the reference point is designated “plus one,”e.g., “+1.”

In some embodiments, the ASOs are complementary to (and bind to) atargeted portion of a SCN1A NIE containing pre-mRNA that is downstream(in the 3′ direction) of the 5′ splice site (or 3′ end of the NIE) ofthe included exon in a SCN1A NIE containing pre-mRNA (e.g., thedirection designated by positive numbers relative to the 5′ splicesite). In some embodiments, the ASOs are complementary to a targetedportion of the SCN1A NIE containing pre-mRNA that is within the regionabout +1 to about +500 relative to the 5′ splice site (or 3′ end) of theincluded exon. In some embodiments, the ASOs may be complementary to atargeted portion of a SCN1A NIE containing pre-mRNA that is within theregion between nucleotides +6 and +496 relative to the 5′ splice site(or 3′ end) of the included exon. In some aspects, the ASOs arecomplementary to a targeted portion that is within the region about +1to about +500, about +1 to about +490, about +1 to about +480, about +1to about +470, about +1 to about +460, about +1 to about +450, about +1to about +440, about +1 to about +430, about +1 to about +420, about +1to about +410, about +1 to about +400, about +1 to about +390, about +1to about +380, about +1 to about +370, about +1 to about +360, about +1to about +350, about +1 to about +340, about +1 to about +330, about +1to about +320, about +1 to about +310, about +1 to about +300, about +1to about +290, about +1 to about +280, about +1 to about +270, about +1to about +260, about +1 to about +250, about +1 to about +240, about +1to about +230, about +1 to about +220, about +1 to about +210, about +1to about +200, about +1 to about +190, about +1 to about +180, about +1to about +170, about +1 to about +160, about +1 to about +150, about +1to about +140, about +1 to about +130, about +1 to about +120, about +1to about +110, about +1 to about +100, about +1 to about +90, about +1to about +80, about +1 to about +70, about +1 to about +60, about +1 toabout +50, about +1 to about +40, about +1 to about +30, or about +1 toabout +20 relative to 5′ splice site (or 3′ end) of the included exon.In some aspects, the ASOs are complementary to a targeted portion thatis within the region from about +1 to about +100, from about +100 toabout +200, from about +200 to about +300, from about +300 to about+400, or from about +400 to about +500 relative to 5′ splice site (or 3′end) of the included exon.

In some embodiments, the ASOs are complementary to (and bind to) atargeted portion of a SCN1A NIE containing pre-mRNA that is upstream (inthe 5′ direction) of the 5′ splice site (or 3′ end) of the included exonin a SCN1A NIE containing pre-mRNA (e.g., the direction designated bynegative numbers relative to the 5′ splice site). In some embodiments,the ASOs are complementary to a targeted portion of the SCN1A NIEcontaining pre-mRNA that is within the region about −4 to about −270relative to the 5′ splice site (or 3′end) of the included exon. In someembodiments, the ASOs may be complementary to a targeted portion of aSCN1A NIE containing pre-mRNA that is within the region betweennucleotides −1 and −264 relative to the 5′ splice site (or 3′ end) ofthe included exon. In some aspects, the ASOs are complementary to atargeted portion that is within the region about −1 to about −270, about−1 to about −260, about −1 to about −250, about −1 to about −240, about−1 to about −230, about −1 to about −220, about −1 to about −210, about−1 to about −200, about −1 to about −190, about −1 to about −180, about−1 to about −170, about −1 to about −160, about −1 to about −150, about−1 to about −140, about −1 to about −130, about −1 to about −120, about−1 to about −110, about −1 to about −100, about −1 to about −90, about−1 to about −80, about −1 to about −70, about −1 to about −60, about −1to about −50, about −1 to about −40, about −1 to about −30, or about −1to about −20 relative to 5′ splice site (or 3′ end) of the includedexon. In some aspects, the ASOs are complementary to a targeted portionthat is within the region from about −1 to about −50, from about −50 toabout −100, from about −100 to about −150, from about −150 to about−200, or from about −200 to about −250 relative to 5′ splice site (or 3′end) of the included exon.

In some embodiments, the ASOs are complementary to a targeted region ofa SCN1A NIE containing pre-mRNA that is upstream (in the 5′ direction)of the 3′ splice site (or 5′ end) of the included exon in a SCN1A NIEcontaining pre-mRNA (e.g., in the direction designated by negativenumbers). In some embodiments, the ASOs are complementary to a targetedportion of the SCN1A NIE containing pre-mRNA that is within the regionabout −1 to about −500 relative to the 3′ splice site (or 5′ end) of theincluded exon. In some embodiments, the ASOs are complementary to atargeted portion of the SCN1A NIE containing pre-mRNA that is within theregion −1 to −496 relative to the 3′ splice site of the included exon.In some aspects, the ASOs are complementary to a targeted portion thatis within the region about −1 to about −500, about −1 to about −490,about −1 to about −480, about −1 to about −470, about −1 to about −460,about −1 to about −450, about −1 to about −440, about −1 to about −430,about −1 to about −420, about −1 to about −410, about −1 to about −400,about −1 to about −390, about −1 to about −380, about −1 to about −370,about −1 to about −360, about −1 to about −350, about −1 to about −340,about −1 to about −330, about −1 to about −320, about −1 to about −310,about −1 to about −300, about −1 to about −290, about −1 to about −280,about −1 to about −270, about −1 to about −260, about −1 to about −250,about −1 to about −240, about −1 to about −230, about −1 to about −220,about −1 to about −210, about −1 to about −200, about −1 to about −190,about −1 to about −180, about −1 to about −170, about −1 to about −160,about −1 to about −150, about −1 to about −140, about −1 to about −130,about −1 to about −120, about −1 to about −110, about −1 to about −100,about −1 to about −90, about −1 to about −80, about −1 to about −70,about −1 to about −60, about −1 to about −50, about −1 to about −40, orabout −1 to about −30 relative to 3′ splice site of the included exon.In some aspects, the ASOs are complementary to a targeted portion thatis within the region from about −1 to about −100, from about −100 toabout −200, from about −200 to about −300, from about −300 to about−400, or from about −400 to about −500 relative to 3′ splice site of theincluded exon.

In some embodiments, the ASOs are complementary to a targeted region ofa SCN1A NIE containing pre-mRNA that is downstream (in the 3′ direction)of the 3′ splice site (5′ end) of the included exon in a SCN1A NIEcontaining pre-mRNA (e.g., in the direction designated by positivenumbers). In some embodiments, the ASOs are complementary to a targetedportion of the SCN1A NIE containing pre-mRNA that is within the regionof about +1 to about +100 relative to the 3′ splice site of the includedexon. In some aspects, the ASOs are complementary to a targeted portionthat is within the region about +1 to about +90, about +1 to about +80,about +1 to about +70, about +1 to about +60, about +1 to about +50,about +1 to about +40, about +1 to about +30, about +1 to about +20, orabout +1 to about +10 relative to 3′ splice site of the included exon.

In some embodiments, the targeted portion of the SCN1A NIE containingpre-mRNA is within the region +100 relative to the 5′ splice site (3′end) of the included exon to −100 relative to the 3′ splice site (5′end) of the included exon. In some embodiments, the targeted portion ofthe SCN1A NIE containing pre-mRNA is within the NIE. In someembodiments, the targeted portion of the SCN1A NIE containing pre-mRNAcomprises a pseudo-exon and intron boundary.

The ASOs may be of any length suitable for specific binding andeffective enhancement of splicing. In some embodiments, the ASOs consistof 8 to 50 nucleobases. For example, the ASO may be 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In someembodiments, the ASOs consist of more than 50 nucleobases. In someembodiments, the ASO is from 8 to 50 nucleobases, 8 to 40 nucleobases, 8to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 13 to 50nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30nucleobases, 13 to 25 nucleobases, 13 to 20 nucleobases, 14 to 50nucleobases, 14 to 40 nucleobases, 14 to 35 nucleobases, 14 to 30nucleobases, 14 to 25 nucleobases, 14 to 20 nucleobases, 15 to 50nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 20 to 50nucleobases, 20 to 40 nucleobases, 20 to 35 nucleobases, 20 to 30nucleobases, 20 to 25 nucleobases, 25 to 50 nucleobases, 25 to 40nucleobases, 25 to 35 nucleobases, or 25 to 30 nucleobases in length. Insome embodiments, the ASOs are 18 nucleotides in length. In someembodiments, the ASOs are 15 nucleotides in length. In some embodiments,the ASOs are 25 nucleotides in length.

In some embodiments, two or more ASOs with different chemistries butcomplementary to the same targeted portion of the NIE containingpre-mRNA are used. In some embodiments, two or more ASOs that arecomplementary to different targeted portions of the NIE containingpre-mRNA are used.

In embodiments, the antisense oligonucleotides of the invention arechemically linked to one or more moieties or conjugates, e.g., atargeting moiety or other conjugate that enhances the activity orcellular uptake of the oligonucleotide. Such moieties include, but arenot limited to, a lipid moiety, e.g., as a cholesterol moiety, acholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecylresidues, a polyamine or a polyethylene glycol chain, or adamantaneacetic acid. Oligonucleotides comprising lipophilic moieties andpreparation methods have been described in the published literature. Inembodiments, the antisense oligonucleotide is conjugated with a moietyincluding, but not limited to, an abasic nucleotide, a polyether, apolyamine, a polyamide, a peptides, a carbohydrate, e.g.,N-acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose(e.g., mannose-6-phosphate), a lipid, or a polyhydrocarbon compound.Conjugates can be linked to one or more of any nucleotides comprisingthe antisense oligonucleotide at any of several positions on the sugar,base or phosphate group, as understood in the art and described in theliterature, e.g., using a linker. Linkers can include a bivalent ortrivalent branched linker. In embodiments, the conjugate is attached tothe 3′ end of the antisense oligonucleotide. Methods of preparingoligonucleotide conjugates are described, e.g., in U.S. Pat. No.8,450,467, “Carbohydrate conjugates as delivery agents foroligonucleotides,” incorporated by reference herein.

In some embodiments, the nucleic acid to be targeted by an ASO is aSCN1A NIE containing pre-mRNA expressed in a cell, such as a eukaryoticcell. In some embodiments, the term “cell” may refer to a population ofcells. In some embodiments, the cell is in a subject. In someembodiments, the cell is isolated from a subject. In some embodiments,the cell is ex vivo. In some embodiments, the cell is a condition ordisease-relevant cell or a cell line. In some embodiments, the cell isin vitro (e.g., in cell culture).

Pharmaceutical Compositions

Pharmaceutical compositions or formulations comprising the agent, e.g.,antisense oligonucleotide, of the described compositions and for use inany of the described methods can be prepared according to conventionaltechniques well known in the pharmaceutical industry and described inthe published literature. In embodiments, a pharmaceutical compositionor formulation for treating a subject comprises an effective amount ofany antisense oligomer as described herein, or a pharmaceuticallyacceptable salt, solvate, hydrate or ester thereof. The pharmaceuticalformulation comprising an antisense oligomer may further comprise apharmaceutically acceptable excipient, diluent or carrier.

Pharmaceutically acceptable salts are suitable for use in contact withthe tissues of humans and lower animals without undue toxicity,irritation, allergic response, etc., and are commensurate with areasonable benefit/risk ratio. (See, e.g., S. M. Berge, et al., J.Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein byreference for this purpose. The salts can be prepared in situ during thefinal isolation and purification of the compounds, or separately byreacting the free base function with a suitable organic acid. Examplesof pharmaceutically acceptable, nontoxic acid addition salts are saltsof an amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother documented methodologies such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate and aryl sulfonate.

In embodiments, the compositions are formulated into any of manypossible dosage forms such as, but not limited to, tablets, capsules,gel capsules, liquid syrups, soft gels, suppositories, and enemas. Inembodiments, the compositions are formulated as suspensions in aqueous,non-aqueous or mixed media. Aqueous suspensions may further containsubstances that increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers. In embodiments, apharmaceutical formulation or composition of the present inventionincludes, but is not limited to, a solution, emulsion, microemulsion,foam or liposome-containing formulation (e.g., cationic or noncationicliposomes).

The pharmaceutical composition or formulation described herein maycomprise one or more penetration enhancers, carriers, excipients orother active or inactive ingredients as appropriate and well known tothose of skill in the art or described in the published literature. Inembodiments, liposomes also include sterically stabilized liposomes,e.g., liposomes comprising one or more specialized lipids. Thesespecialized lipids result in liposomes with enhanced circulationlifetimes. In embodiments, a sterically stabilized liposome comprisesone or more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. In embodiments, asurfactant is included in the pharmaceutical formulation orcompositions. The use of surfactants in drug products, formulations andemulsions is well known in the art. In embodiments, the presentinvention employs a penetration enhancer to effect the efficientdelivery of the antisense oligonucleotide, e.g., to aid diffusion acrosscell membranes and/or enhance the permeability of a lipophilic drug. Inembodiments, the penetration enhancers are a surfactant, fatty acid,bile salt, chelating agent, or non-chelating nonsurfactant.

In embodiments, the pharmaceutical formulation comprises multipleantisense oligonucleotides. In embodiments, the antisenseoligonucleotide is administered in combination with another drug ortherapeutic agent.

Combination Therapies

In some embodiments, the ASOs disclosed in the present disclosure can beused in combination with one or more additional therapeutic agents. Insome embodiments, the one or more additional therapeutic agents cancomprise a small molecule. For example, the one or more additionaltherapeutic agents can comprise a small molecule described inWO2016128343A1, WO2017053982A1, WO2016196386A1, WO201428459A1,WO201524876A2, WO2013119916A2, and WO2014209841A2, which areincorporated by reference herein in their entirety. In some embodiments,the one or more additional therapeutic agents comprise an ASO that canbe used to correct intron retention. In some embodiments, the one ormore other agents are selected from the ASOs listed in Table 1a or Table1b.

TABLE 1a Exemplary ASOs to correct intron retention SEQ Retained ID NO:Name Sequence (5′-3′) Intron 115 SCN1A-IVS21+6 CAGAGAAAAUAGUGUUCA 21 116SCN1A-IVS21+11 AUAUUCAGAGAAAAUAGU 21 117 SCN1A-IVS21+16UAAAAAUAUUCAGAGAAA 21 118 SCN1A-IVS21+21 AACAAUAAAAAUAUUCAG 21 119SCN1A-IVS21+26 UUCCAAACAAUAAAAAUA 21 120 SCN1A-IVS21+31UAUUAUUCCAAACAAUAA 21 121 SCN1A-IVS21+36 UUUGUUAUUAUUCCAAAC 21 122SCN1A-IVS21+41 AUUAUUUUGUUAUUAUUC 21 123 SCN1A-IVS21+46AUGUCAUUAUUUUGUUAU 21 124 SCN1A-IVS21+51 GAUGUAUGUCAUUAUUUU 21 125SCN1A-IVS21+56 UAAUAGAUGUAUGUCAUU 21 126 SCN1A-IVS21+61CUAAAUAAUAGAUGUAUG 21 127 SCN1A-IVS21+66 AGGAACUAAAUAAUAGAU 21 128SCN1A-IVS21+71 UUCUUAGGAACUAAAUAA 21 129 SCN1A-IVS21+76ACUUUUUCUUAGGAACUA 21 130 SCN1A-IVS21+81 UAUAUACUUUUUCUUAGG 21 131SCN1A-IVS21−16 UGCAUGUUUUACUUUGGA 21 132 SCN1A-IVS21−21GUUUUACUUUGGAGUAAA 21 133 SCN1A-IVS21−26 ACUUUGGAGUAAAAAUAA 21 134SCN1A-IVS21−31 GGAGUAAAAAUAAUUUAG 21 135 SCN1A-IVS21−36AAAAAUAAUUUAGACCUG 21 136 SCN1A-IVS21−41 UAAUUUAGACCUGAUGUU 21 137SCN1A-IVS21−46 UAGACCUGAUGUUUAAUA 21 138 SCN1A-IVS21−51CUGAUGUUUAAUAAAUAU 21 139 SCN1A-IVS21−56 GUUUAAUAAAUAUUCUUA 21 140SCN1A-IVS21−61 AUAAAUAUUCUUACUGAU 21 141 SCN1A-IVS21−66UAUUCUUACUGAUAUAAU 21 142 SCN1A-IVS21−71 UUACUGAUAUAAUUUUCA 21 143SCN1A-IVS21−76 GAUAUAAUUUUCAAAAGG 21 144 SCN1A-IVS21−81AAUUUUCAAAAGGGAAUA 21 145 SCN1A-IVS21−27 CUUUGGAGUAAAAAUAAU 21 146SCN1A-IVS21−28 UUUGGAGUAAAAAUAAUU 21 148 SCN1A-IVS21−29UUGGAGUAAAAAUAAUUU 21 149 SCN1A-IVS21−30 UGGAGUAAAAAUAAUUUA 21 150SCN1A-IVS21−32 GAGUAAAAAUAAUUUAGA 21 151 SCN1A-IVS21−33AGUAAAAAUAAUUUAGAC 21 152 SCN1A-IVS21−34 GUAAAAAUAAUUUAGACC 21 153SCN1A-IVS21−35 UAAAAAUAAUUUAGACCU 21 154 SCN1A-IVS21−72UACUGAUAUAAUUUUCAA 21 155 SCN1A-IVS21−73 ACUGAUAUAAUUUUCAAA 21 156SCN1A-IVS21−74 CUGAUAUAAUUUUCAAAA 21 157 SCN1A-IVS21−75UGAUAUAAUUUUCAAAAG 21 158 SCN1A-IVS21−77 AUAUAAUUUUCAAAAGGG 21 159SCN1A-IVS21−78 UAUAAUUUUCAAAAGGGA 21 160 SCN1A-IVS21−79AUAAUUUUCAAAAGGGAA 21 161 SCN1A-IVS21−80 UAAUUUUCAAAAGGGAAU 21 162CAAGGAUUAAAGGUAGCA 21

TABLE 1b Exemplary ASOs to correct intron retention SEQ Retained ID NO:Name SeqTence (5′-3′) Intron 163 SCN1A-IVS21+6 CAGAGAAAATAGTGTTCA 21 164SCN1A-IVS21+11 ATATTCAGAGAAAATAGT 21 165 SCN1A-IVS21+16TAAAAATATTCAGAGAAA 21 166 SCN1A-IVS21+21 AACAATAAAAATATTCAG 21 167SCN1A-IVS21+26 TTCCAAACAATAAAAATA 21 168 SCN1A-IVS21+31TATTATTCCAAACAATAA 21 169 SCN1A-IVS21+36 TTTGTTATTATTCCAAAC 21 170SCN1A-IVS21+41 ATTATTTTGTTATTATTC 21 171 SCN1A-IVS21+46ATGTCATTATTTTGTTAT 21 172 SCN1A-IVS21+51 GATGTATGTCATTATTTT 21 173SCN1A-IVS21+56 TAATAGATGTATGTCATT 21 174 SCN1A-IVS21+61CTAAATAATAGATGTATG 21 175 SCN1A-IVS21+66 AGGAACTAAATAATAGAT 21 176SCN1A-IVS21+71 TTCTTAGGAACTAAATAA 21 177 SCN1A-IVS21+76ACTTTTTCTTAGGAACTA 21 178 SCN1A-IVS21+81 TATATACTTTTTCTTAGG 21 179SCN1A-IVS21−16 TGCATGTTTTACTTTGGA 21 180 SCN1A-IVS21−21GTTTTACTTTGGAGTAAA 21 181 SCN1A-IVS21−26 ACTTTGGAGTAAAAATAA 21 182SCN1A-IVS21−31 GGAGTAAAAATAATTTAG 21 183 SCN1A-IVS21−36AAAAATAATTTAGACCTG 21 184 SCN1A-IVS21−41 TAATTTAGACCTGATGTT 21 185SCN1A-IVS21−46 TAGACCTGATGTTTAATA 21 186 SCN1A-IVS21−51CTGATGTTTAATAAATAT 21 187 SCN1A-IVS21−56 GTTTAATAAATATTCTTA 21 188SCN1A-IVS21−61 ATAAATATTCTTACTGAT 21 189 SCN1A-IVS21−66TATTCTTACTGATATAAT 21 190 SCN1A-IVS21−71 TTACTGATATAATTTTCA 21 191SCN1A-IVS21−76 GATATAATTTTCAAAAGG 21 192 SCN1A-IVS21−81AATTTTCAAAAGGGAATA 21 193 SCN1A-IVS21−27 CTTTGGAGTAAAAATAAT 21 194SCN1A-IVS21−28 TTTGGAGTAAAAATAATT 21 195 SCN1A-IVS21−29TTGGAGTAAAAATAATTT 21 196 SCN1A-IVS21−30 TGGAGTAAAAATAATTTA 21 197SCN1A-IVS21−32 GAGTAAAAATAATTTAGA 21 198 SCN1A-IVS21−33AGTAAAAATAATTTAGAC 21 199 SCN1A-IVS21−34 GTAAAAATAATTTAGACC 21 200SCN1A-IVS21−35 TAAAAATAATTTAGACCT 21 201 SCN1A-IVS21−72TACTGATATAATTTTCAA 21 202 SCN1A-IVS21−73 ACTGATATAATTTTCAAA 21 203SCN1A-IVS21−74 CTGATATAATTTTCAAAA 21 204 SCN1A-IVS21−75TGATATAATTTTCAAAAG 21 205 SCN1A-IVS21−77 ATATAATTTTCAAAAGGG 21 206SCN1A-IVS21−78 TATAATTTTCAAAAGGGA 21 207 SCN1A-IVS21−79ATAATTTTCAAAAGGGAA 21 208 SCN1A-IVS21−80 TAATTTTCAAAAGGGAAT 21 209CAAGGATTAAAGGTAGCA 21Treatment of Subjects

Any of the compositions provided herein may be administered to anindividual. “Individual” may be used interchangeably with “subject” or“patient.” An individual may be a mammal, for example a human or animalsuch as a non-human primate, a rodent, a rabbit, a rat, a mouse, ahorse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep. Inembodiments, the individual is a human. In embodiments, the individualis a fetus, an embryo, or a child. In other embodiments, the individualmay be another eukaryotic organism, such as a plant. In someembodiments, the compositions provided herein are administered to a cellex vivo.

In some embodiments, the compositions provided herein are administeredto an individual as a method of treating a disease or disorder. In someembodiments, the individual has a genetic disease, such as any of thediseases described herein. In some embodiments, the individual is atrisk of having a disease, such as any of the diseases described herein.In some embodiments, the individual is at increased risk of having adisease or disorder caused by insufficient amount of a protein orinsufficient activity of a protein. If an individual is “at an increasedrisk” of having a disease or disorder caused insufficient amount of aprotein or insufficient activity of a protein, the method involvespreventative or prophylactic treatment. For example, an individual maybe at an increased risk of having such a disease or disorder because offamily history of the disease. Typically, individuals at an increasedrisk of having such a disease or disorder benefit from prophylactictreatment (e.g., by preventing or delaying the onset or progression ofthe disease or disorder). In embodiments, a fetus is treated in utero,e.g., by administering the ASO composition to the fetus directly orindirectly (e.g., via the mother).

Suitable routes for administration of ASOs of the present invention mayvary depending on cell type to which delivery of the ASOs is desired.Multiple tissues and organs are affected by Dravet syndrome; Epilepsy,generalized, with febrile seizures plus, type 2; Febrile seizures,familial, 3A; Migraine, familial hemiplegic, 3; Autism; Epilepticencephalopathy, early infantile, 13; Sick sinus syndrome 1; Alzheimer'sdisease or SUDEP, with the brain being the most significantly affectedtissue. The ASOs of the present invention may be administered topatients parenterally, for example, by intrathecal injection,intracerebroventricular injection, intraperitoneal injection,intramuscular injection, subcutaneous injection, intravitreal injection,or intravenous injection.

In some embodiments, the disease or condition is induced by a mutationin Na_(v)1.1 (a protein encoded by the SCN1A gene). In some instances,the mutation is a loss-of-function mutation in Na_(v)1.1. In some cases,the loss-of-function mutation in Na_(v)1.1 comprises one or moremutations that decreases or impairs the function of Na_(v)1.1 (e.g., by10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) relative tothe function of a wild-type Na_(v)1.1. In some cases, theloss-of-function mutation in Na_(v)1.1 comprises one or more mutationsthat result in a disease phenotype. Exemplary loss-of-function mutationsinclude, but are not limited to, R859C, T875M, V1353L, I1656M, R1657C,A1685V, M1841T, and R1916G.

In other instances, the mutation is a gain-of-function mutation inNav1.1. In such cases, the gain-of-function mutation comprises one ormore mutations that prolongs activation of Na_(v)1.1 (e.g., by 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) relative to thefunction of a wild-type Na_(v)1.1. In such cases, the gain-of-functionmutation in Na_(v)1.1 comprises one or more mutations that result in adisease phenotype. Exemplary gain-of-function mutations include, but arenot limited to, D188V, W1204R, R1648H, and D1866Y.

In some embodiments, the disease or condition is an encephalopathy. Insome cases, the encephalopathy is induced by a loss-of-function mutationin Nav1.1.

In some embodiments, the encephalopathy is epileptic encephalopathy.Exemplary epileptic encephalopathies include, but are not limited to,Dravet Syndrome (DS) (also known as severe myoclonic epilepsy of infancyor SMEI); severe myoclonic epilepsy of infancy (SMEI)-borderland (SMEB);Febrile seizure (FS); epilepsy, generalized, with febrile seizures plus(GEFS+); epileptic encephalopathy, early infantile, 13; cryptogenicgeneralized epilepsy; cryptogenic focal epilepsy; myoclonic-astaticepilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms;early myoclonic encephalopathy; progressive myoclonic epilepsy;alternating hemiplegia of childhood; unclassified epilepticencephalopathy; sudden unexpected death in epilepsy (SUDEP); earlyinfantile SCN1A encephalopathy; early infantile epileptic encephalopathy(EIEE); or sick sinus syndrome 1. In some embodiments, the disease orcondition is epileptic encephalopathy, optionally selected from DravetSyndrome (DS) (also known as severe myoclonic epilepsy of infancy orSMEI); severe myoclonic epilepsy of infancy (SMEI)-borderland (SMEB);Febrile seizure (FS); epilepsy, generalized, with febrile seizures plus(GEFS+); epileptic encephalopathy, early infantile, 13; cryptogenicgeneralized epilepsy; cryptogenic focal epilepsy; myoclonic-astaticepilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms;early myoclonic encephalopathy; progressive myoclonic epilepsy;alternating hemiplegia of childhood; unclassified epilepticencephalopathy; sudden unexpected death in epilepsy (SUDEP); and sicksinus syndrome 1.

In some instances, GEFS+ is epilepsy, generalized, with febrile seizuresplus, type 2.

In some instances, the Febrile seizure is Febrile seizures, familial,3A.

In some instances, SMEB is SMEB without generalized spike wave(SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB lacking morethan one feature of SMEI (SMEB-O), or intractable childhood epilepsywith generalized tonic-clonic seizures (ICEGTC).

In some embodiments, the diseases or conditions induced by aloss-of-function mutation in Na_(v)1.1 include, but are not limited to,Dravet Syndrome (DS) (also known as SMEI); severe myoclonic epilepsy ofinfancy (SMEI)-borderland (SMEB); Febrile seizure (FS); epilepsy,generalized, with febrile seizures plus (GEFS+); epilepticencephalopathy, early infantile, 13; cryptogenic generalized epilepsy;cryptogenic focal epilepsy; myoclonic-astatic epilepsy; Lennox-Gastautsyndrome; West syndrome; idiopathic spasms; early myoclonicencephalopathy; progressive myoclonic epilepsy; alternating hemiplegiaof childhood; unclassified epileptic encephalopathy; sudden unexpecteddeath in epilepsy (SUDEP); sick sinus syndrome 1; early infantile SCN1Aencephalopathy; early infantile epileptic encephalopathy (EIEE); autism;or malignant migrating partial seizures of infancy.

In some embodiments, the disease or condition is induced by again-of-function mutation in Na_(v)1.1. Exemplary diseases or conditionsassociated with a gain-of-function mutation in Na_(v)1.1 include, butare not limited to, migraine. In some instances, the disease orcondition induced by a gain-of-function mutation in Na_(v)1.1 ismigraine.

In some instances, the migraine is migraine, familial hemiplegic, 3.

In some embodiments, the disease or condition is a Na_(v)1.1 geneticepilepsy. The Nav1.1 genetic epilepsy can include a loss-of-functionmutation in Na_(v)1.1 or a gain-of-function mutation in Na_(v)1.1. Insome cases, the Na_(v)1.1 genetic epilepsy includes one or morehereditary mutations. In other cases, the Na_(v)1.1 genetic epilepsyincludes one or more de novo mutations. In some cases, the Na_(v)1.1genetic epilepsy includes Dravet Syndrome (DS) (also known as severemyoclonic epilepsy of infancy or SMEI); severe myoclonic epilepsy ofinfancy (SMEI)-borderland (SMEB); Febrile seizure (FS); epilepsy,generalized, with febrile seizures plus (GEFS+); epilepticencephalopathy, early infantile, 13; cryptogenic generalized epilepsy;cryptogenic focal epilepsy; myoclonic-astatic epilepsy; Lennox-Gastautsyndrome; West syndrome; idiopathic spasms; early myoclonicencephalopathy; progressive myoclonic epilepsy; alternating hemiplegiaof childhood; unclassified epileptic encephalopathy; early infantileSCN1A encephalopathy; early infantile epileptic encephalopathy (EIEE);sudden unexpected death in epilepsy (SUDEP); or malignant migratingpartial seizures of infancy. In some cases, the Na_(v)1.1 geneticepilepsy associated with a loss-of-function mutation in Na_(v)1.1includes Dravet Syndrome (DS) (also known as severe myoclonic epilepsyof infancy or SMEI); severe myoclonic epilepsy of infancy(SMEI)-borderland (SMEB); Febrile seizure (FS); epilepsy, generalized,with febrile seizures plus (GEFS+); epileptic encephalopathy, earlyinfantile, 13; cryptogenic generalized epilepsy; cryptogenic focalepilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; Westsyndrome; idiopathic spasms; early myoclonic encephalopathy; progressivemyoclonic epilepsy; alternating hemiplegia of childhood; unclassifiedepileptic encephalopathy; early infantile SCN1A encephalopathy; earlyinfantile epileptic encephalopathy (EIEE); sudden unexpected death inepilepsy (SUDEP); malignant migrating partial seizures of infancy.

In some embodiments, the disease or condition is associated with ahaploinsufficiency of the SCN1A gene. Exemplary diseases or conditionsassociated with a haploinsufficiency of the SCN1A gene include, but arenot limited to, Dravet Syndrome (DS) (also known as SMEI); severemyoclonic epilepsy of infancy (SMEI)-borderland (SMEB); Febrile seizure(FS); epilepsy, generalized, with febrile seizures plus (GEFS+);epileptic encephalopathy, early infantile, 13; cryptogenic generalizedepilepsy; cryptogenic focal epilepsy; myoclonic-astatic epilepsy;Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; earlymyoclonic encephalopathy; progressive myoclonic epilepsy; alternatinghemiplegia of childhood; unclassified epileptic encephalopathy; suddenunexpected death in epilepsy (SUDEP); sick sinus syndrome 1; earlyinfantile SCN1A encephalopathy; early infantile epileptic encephalopathy(EIEE); or malignant migrating partial seizures of infancy. In somecases, the disease or condition is Dravet Syndrome (DS) (also known asSMEI); severe myoclonic epilepsy of infancy (SMEI)-borderland (SMEB);Febrile seizure (FS); epilepsy, generalized, with febrile seizures plus(GEFS+); epileptic encephalopathy, early infantile, 13; cryptogenicgeneralized epilepsy; cryptogenic focal epilepsy; myoclonic-astaticepilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms;early myoclonic encephalopathy; progressive myoclonic epilepsy;alternating hemiplegia of childhood; unclassified epilepticencephalopathy; sudden unexpected death in epilepsy (SUDEP); sick sinussyndrome 1; early infantile SCN1A encephalopathy; early infantileepileptic encephalopathy (EIEE); or malignant migrating partial seizuresof infancy.

In some cases, the disease or condition is Dravet Syndrome (DS).

Dravet syndrome (DS), otherwise known as severe myoclonic epilepsy ofinfancy (SMEI), is an epileptic encephalopathy presenting in the firstyear of life. Dravet syndrome is an increasingly recognized epilepticencephalopathy in which the clinical diagnosis is supported by thefinding of sodium channel gene mutations in approximately 70-80% ofpatients. Mutations of ion channel genes play a major role in thepathogenesis of a range of epilepsy syndromes, resulting in someepilepsies being regarded as channelopathies. Voltage-gated sodiumchannels (VGSCs) play an essential role in neuronal excitability;therefore, it is not surprising that many mutations associated with DShave been identified in the gene encoding a VGSC subunit. The disease isdescribed by, e.g., Mulley, et al., 2005, and the disease description atOMIM #607208 (Online Mendelian Inheritance in Man, Johns HopkinsUniversity, 1966-2015), both incorporated by reference herein.

Between 70% and 80% of patients carry sodium channel al subunit gene(SCN1A) abnormalities, and truncating mutations account for about 40%,and have a significant correlation with an earlier age of seizuresonset. Sequencing mutations are found in about 70% of cases and comprisetruncating (40%) and missense mutations (40%) with the remaining beingsplice-site changes. Most mutations are de novo, but familial mutationsoccur in 5-10% of cases and are usually missense in nature. Theremaining SCN1A mutations comprise splice-site and missense mutations,most of which fall into the pore-forming region of the sodium channel.At present, over 500 mutations have been associated with DS and arerandomly distributed along the gene (Mulley, et al., Neurol. 2006, 67,1094-1095).

The SCN1A gene is located in the cluster of sodium channel genes onhuman chromosome 2q24 and encodes the α-pore forming subunits known asNa_(v)1.1 of the neuronal voltage gated sodium channel. The SCN1A genespans approximately 100 kb of genomic DNA and comprises 26 exons. TheSCN1A protein consists of four domains, each with six-transmembranesegments. Two splice variants have been identified that result in a longand short isoform that differ in the presence or absence of 11 aminoacids in the cytoplasmic loop between domains 1 and 2, in exon 11(Miller, et al., 1993-2015, and Mulley, et al., 2005, 25, 535-542,incorporated herein by reference).

Alternative splicing events in SCN1A gene can lead to non-productivemRNA transcripts which in turn can lead to aberrant protein expression,and therapeutic agents which can target the alternative splicing eventsin SCN1A gene can modulate the expression level of functional proteinsin DS patients and/or inhibit aberrant protein expression. Suchtherapeutic agents can be used to treat a condition caused by SCN1Aprotein deficiency.

One of the alternative splicing events that can lead to non-productivemRNA transcripts is the inclusion of an extra exon in the mRNAtranscript that can induce non-sense mediated mRNA decay. The presentdisclosure provides compositions and methods for modulating alternativesplicing of SCN1A to increase the production of protein-coding maturemRNA, and thus, translated functional SCN1A protein. These compositionsand methods include antisense oligomers (ASOs) that can cause exonskipping and promote constitutive splicing of SCN1A pre-mRNA. In variousembodiments, functional SCN1A protein can be increased using the methodsof the disclosure to treat a condition caused by SCN1A proteindeficiency.

In some cases, the disease or condition is SMEB.

In some cases, the disease or condition is GEFS+.

In some cases, the disease or condition is a Febrile seizure (e.g.,Febrile seizures, familial, 3A).

In some cases, the disease or condition is autism (also known as autismspectrum disorder or ASD).

In some cases, the disease or condition is migraine (e.g., migraine,familial hemiplegic, 3).

In some cases, the disease or condition is Alzheimer's disease.

In some embodiments, the disease or condition is SCN2A encephalopathy.

In some embodiments, the disease or condition is SCN8A encephalopathy.

In some embodiments, the disease or condition is SCN5A arrhythmia.

In embodiments, the antisense oligonucleotide is administered with oneor more agents capable of promoting penetration of the subject antisenseoligonucleotide across the blood-brain barrier by any method known inthe art. For example, delivery of agents by administration of anadenovirus vector to motor neurons in muscle tissue is described in U.S.Pat. No. 6,632,427, “Adenoviral-vector-mediated gene transfer intomedullary motor neurons,” incorporated herein by reference. Delivery ofvectors directly to the brain, e.g., the striatum, the thalamus, thehippocampus, or the substantia nigra, is described, e.g., in U.S. Pat.No. 6,756,523, “Adenovirus vectors for the transfer of foreign genesinto cells of the central nervous system particularly in brain,”incorporated herein by reference.

In embodiments, the antisense oligonucleotides are linked or conjugatedwith agents that provide desirable pharmaceutical or pharmacodynamicproperties. In embodiments, the antisense oligonucleotide is coupled toa substance, known in the art to promote penetration or transport acrossthe blood-brain barrier, e.g., an antibody to the transferrin receptor.In embodiments, the antisense oligonucleotide is linked with a viralvector, e.g., to render the antisense compound more effective orincrease transport across the blood-brain barrier. In embodiments,osmotic blood brain barrier disruption is assisted by infusion ofsugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose,D(+) xylose, dulcitol, myo-inositol, L(−) fructose, D(−) mannitol, D(+)glucose, D(+) arabinose, D(−) arabinose, cellobiose, D(+) maltose, D(+)raffinose, L(+) rhamnose, D(+) melibiose, D(−) ribose, adonitol, D(+)arabitol, L(−) arabitol, D(+) fucose, L(−) fucose, D(−) lyxose, L(+)lyxose, and L(−) lyxose, or amino acids, e.g., glutamine, lysine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine,histidine, leucine, methionine, phenylalanine, proline, serine,threonine, tyrosine, valine, and taurine. Methods and materials forenhancing blood brain barrier penetration are described, e.g., in U.S.Pat. No. 9,193,969, “Compositions and methods for selective delivery ofoligonucleotide molecules to specific neuron types,” U.S. Pat. No.4,866,042, “Method for the delivery of genetic material across the bloodbrain barrier,” U.S. Pat. No. 6,294,520, “Material for passage throughthe blood-brain barrier,” and U.S. Pat. No. 6,936,589, “Parenteraldelivery systems,” each incorporated herein by reference.

In embodiments, an ASO of the invention is coupled to a dopaminereuptake inhibitor (DRI), a selective serotonin reuptake inhibitor(SSRI), a noradrenaline reuptake inhibitor (NRI), anorepinephrine-dopamine reuptake inhibitor (NDRI), and aserotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI), usingmethods described in, e.g., U.S. Pat. No. 9,193,969, incorporated hereinby reference.

In embodiments, subjects treated using the methods and compositions areevaluated for improvement in condition using any methods known anddescribed in the art.

Methods of Identifying Additional ASOs that Induce Exon Skipping

Also within the scope of the present disclosure are methods foridentifying or determining ASOs that induce exon skipping of a SCN1A NIEcontaining pre-mRNA. For example, a method can comprise identifying ordetermining ASOs that induce pseudo-exon skipping of a SCN1A NIEcontaining pre-mRNA. ASOs that specifically hybridize to differentnucleotides within the target region of the pre-mRNA may be screened toidentify or determine ASOs that improve the rate and/or extent ofsplicing of the target intron. In some embodiments, the ASO may block orinterfere with the binding site(s) of a splicing repressor(s)/silencer.Any method known in the art may be used to identify (determine) an ASOthat when hybridized to the target region of the exon results in thedesired effect (e.g., pseudo-exon skipping, protein or functional RNAproduction). These methods also can be used for identifying ASOs thatinduce exon skipping of the included exon by binding to a targetedregion in an intron flanking the included exon, or in a non-includedexon. An example of a method that may be used is provided below.

A round of screening, referred to as an ASO “walk” may be performedusing ASOs that have been designed to hybridize to a target region of apre-mRNA. For example, the ASOs used in the ASO walk can be tiled every5 nucleotides from approximately 100 nucleotides upstream of the 3′splice site of the included exon (e.g., a portion of sequence of theexon located upstream of the target/included exon) to approximately 100nucleotides downstream of the 3′ splice site of the target/included exonand/or from approximately 100 nucleotides upstream of the 5′ splice siteof the included exon to approximately 100 nucleotides downstream of the5′ splice site of the target/included exon (e.g., a portion of sequenceof the exon located downstream of the target/included exon). Forexample, a first ASO of 15 nucleotides in length may be designed tospecifically hybridize to nucleotides +6 to +20 relative to the 3′splice site of the target/included exon. A second ASO may be designed tospecifically hybridize to nucleotides +11 to +25 relative to the 3′splice site of the target/included exon. ASOs are designed as suchspanning the target region of the pre-mRNA. In embodiments, the ASOs canbe tiled more closely, e.g., every 1, 2, 3, or 4 nucleotides. Further,the ASOs can be tiled from 100 nucleotides downstream of the 5′ splicesite, to 100 nucleotides upstream of the 3′ splice site. In someembodiments, the ASOs can be tiled from about 1,160 nucleotides upstreamof the 3′ splice site, to about 500 nucleotides downstream of the 5′splice site. In some embodiments, the ASOs can be tiled from about 500nucleotides upstream of the 3′ splice site, to about 1,920 nucleotidesdownstream of the 3′ splice site.

One or more ASOs, or a control ASO (an ASO with a scrambled sequence,sequence that is not expected to hybridize to the target region) aredelivered, for example by transfection, into a disease-relevant cellline that expresses the target pre-mRNA (e.g., a NIE containing pre-mRNAdescribed herein). The exon skipping effects of each of the ASOs may beassessed by any method known in the art, for example by reversetranscriptase (RT)-PCR using primers that span the splice junction, asdescribed in Example 4. A reduction or absence of a longer RT-PCRproduct produced using the primers spanning the region containing theincluded exon (e.g. including the flanking exons of the NIE) inASO-treated cells as compared to in control ASO-treated cells indicatesthat splicing of the target NIE has been enhanced. In some embodiments,the exon skipping efficiency (or the splicing efficiency to splice theintron containing the NIE), the ratio of spliced to unspliced pre-mRNA,the rate of splicing, or the extent of splicing may be improved usingthe ASOs described herein. The amount of protein or functional RNA thatis encoded by the target pre-mRNA can also be assessed to determinewhether each ASO achieved the desired effect (e.g., enhanced functionalprotein production). Any method known in the art for assessing and/orquantifying protein production, such as Western blotting, flowcytometry, immunofluorescence microscopy, and ELISA, can be used.

A second round of screening, referred to as an ASO “micro-walk” may beperformed using ASOs that have been designed to hybridize to a targetregion of a pre-mRNA. The ASOs used in the ASO micro-walk are tiledevery 1 nucleotide to further refine the nucleotide acid sequence of thepre-mRNA that when hybridized with an ASO results in exon skipping (orenhanced splicing of NIE).

Regions defined by ASOs that promote splicing of the target intron areexplored in greater detail by means of an ASO “micro-walk”, involvingASOs spaced in 1-nt steps, as well as longer ASOs, typically 18-25 nt.

As described for the ASO walk above, the ASO micro-walk is performed bydelivering one or more ASOs, or a control ASO (an ASO with a scrambledsequence, sequence that is not expected to hybridize to the targetregion), for example by transfection, into a disease-relevant cell linethat expresses the target pre-mRNA. The splicing-inducing effects ofeach of the ASOs may be assessed by any method known in the art, forexample by reverse transcriptase (RT)-PCR using primers that span theNIE, as described herein (see, e.g., Example 4). A reduction or absenceof a longer RT-PCR product produced using the primers spanning the NIEin ASO-treated cells as compared to in control ASO-treated cellsindicates that exon skipping (or splicing of the target introncontaining an NIE) has been enhanced. In some embodiments, the exonskipping efficiency (or the splicing efficiency to splice the introncontaining the NIE), the ratio of spliced to unspliced pre-mRNA, therate of splicing, or the extent of splicing may be improved using theASOs described herein. The amount of protein or functional RNA that isencoded by the target pre-mRNA can also be assessed to determine whethereach ASO achieved the desired effect (e.g., enhanced functional proteinproduction). Any method known in the art for assessing and/orquantifying protein production, such as Western blotting, flowcytometry, immunofluorescence microscopy, and ELISA, can be used.

ASOs that when hybridized to a region of a pre-mRNA result in exonskipping (or enhanced splicing of the intron containing a NIE) andincreased protein production may be tested in vivo using animal models,for example transgenic mouse models in which the full-length human genehas been knocked-in or in humanized mouse models of disease. Suitableroutes for administration of ASOs may vary depending on the diseaseand/or the cell types to which delivery of the ASOs is desired. ASOs maybe administered, for example, by intrathecal injection,intracerebroventricular injection, intraperitoneal injection,intramuscular injection, subcutaneous injection, intravitreal injection,or intravenous injection. Following administration, the cells, tissues,and/or organs of the model animals may be assessed to determine theeffect of the ASO treatment by for example evaluating splicing(efficiency, rate, extent) and protein production by methods known inthe art and described herein. The animal models may also be anyphenotypic or behavioral indication of the disease or disease severity.

As described herein in various examples, exon 20x in human SCN1A gene isequivalent to exon 21x in mouse SCN1A gene.

Also within the scope of the present disclosure is a method to identifyor validate an NMD-inducing exon in the presence of an NMD inhibitor,for example, cycloheximide. An exemplary method is provided in FIG. 3and Example 2.

SPECIFIC EMBODIMENTS Embodiment 1

A method of modulating expression of SCN1A protein in a cell having anmRNA that contains a non-sense mediated RNA decay-inducing exon (NMDexon mRNA) and encodes SCN1A protein, the method comprising contacting atherapeutic agent to the cell, whereby the therapeutic agent modulatessplicing of the NMD exon from the NMD exon mRNA encoding SCN1A protein,thereby modulating the level of processed mRNA encoding SCN1A protein,and modulating expression of SCN1A protein in the cell.

Embodiment 2

A method of treating a disease or condition in a subject in need thereofby modulating expression of SCN1A protein in a cell of the subject,comprising: contacting the cell of the subject with a therapeutic agentthat modulates splicing of a non-sense mediated mRNA decay-inducing exon(NMD exon) from an mRNA in the cell that contains the NMD exon andencodes SCN1A, thereby modulating the level of processed mRNA encodingthe SCN1A protein, and modulating expression of SCN1A protein in thecell of the subject.

Embodiment 3

The method of embodiment 1 or 2, wherein the therapeutic agent

(a) binds to a targeted portion of the NMD exon mRNA encoding SCN1A;

(b) modulates binding of a factor involved in splicing of the NMD exonmRNA; or

(c) a combination of (a) and (b).

Embodiment 4

The method of embodiment 3, wherein the therapeutic agent interfereswith binding of the factor involved in splicing of the NMD exon from aregion of the targeted portion.

Embodiment 5

The method of embodiment 3 or 4, wherein the targeted portion isproximal to the NMD exon.

Embodiment 6

The method of any one of embodiments 3 to 5, wherein the targetedportion is at most about 1500 nucleotides, about 1000 nucleotides, about800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500nucleotides, about 400 nucleotides, about 300 nucleotides, about 200nucleotides, about 100 nucleotides, about 80 nucleotides, about 70nucleotides, about 60 nucleotides, about 50 nucleotides upstream of 5′end of the NMD exon.

Embodiment 7

The method of any one of embodiments 3 to 6, wherein the targetedportion is at least about 1500 nucleotides, about 1000 nucleotides,about 800 nucleotides, about 700 nucleotides, about 600 nucleotides,about 500 nucleotides, about 400 nucleotides, about 300 nucleotides,about 200 nucleotides, about 100 nucleotides, about 80 nucleotides,about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10nucleotides, about 5 nucleotides, about 4 nucleotides, about 2nucleotides, about 1 nucleotides upstream of 5′ end of the NMD exon.

Embodiment 8

The method of any one of embodiments 3 to 5, wherein the targetedportion is at most about 1500 nucleotides, about 1000 nucleotides, about800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500nucleotides, about 400 nucleotides, about 300 nucleotides, about 200nucleotides, about 100 nucleotides, about 80 nucleotides, about 70nucleotides, about 60 nucleotides, about 50 nucleotides downstream of 3′end of the NMD exon.

Embodiment 9

The method of any one of embodiments 3 to 5 or 8, wherein the targetedportion is at least about 1500 nucleotides, about 1000 nucleotides,about 800 nucleotides, about 700 nucleotides, about 600 nucleotides,about 500 nucleotides, about 400 nucleotides, about 300 nucleotides,about 200 nucleotides, about 100 nucleotides, about 80 nucleotides,about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10nucleotides, about 5 nucleotides, about 4 nucleotides, about 2nucleotides, about 1 nucleotides downstream of 3′ end of the NMD exon.

Embodiment 10

The method of any one of embodiments 3 to 9, wherein the targetedportion is located in an intronic region between two canonical exonicregions of the NMD exon mRNA encoding SCN1A, and wherein the intronicregion contains the NMD exon.

Embodiment 11

The method of any one of embodiments 3 to 10, wherein the targetedportion at least partially overlaps with the NMD exon.

Embodiment 12

The method of any one of embodiments 3 to 11, wherein the targetedportion at least partially overlaps with an intron upstream of the NMDexon.

Embodiment 13

The method of any one of embodiments 3 to 12, wherein the targetedportion comprises 5′ NMD exon-intron junction or 3′ NMD exon-intronjunction.

Embodiment 14

The method of any one of embodiments 3 to 13, wherein the targetedportion is within the NMD exon.

Embodiment 15

The method of any one of embodiments 3 to 14, wherein the targetedportion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutivenucleotides of the NMD exon.

Embodiment 16

The method of any one of embodiments 1 to 15, wherein the NMD exon mRNAencoding SCN1A comprises a sequence with at least about 80%, 85%, 90%,95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 2 or 7-10.

Embodiment 17

The method of any one of embodiments 1 to 16, wherein the NMD exon mRNAencoding SCN1A is encoded by a genetic sequence with at least about 80%,85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NOs: 1 or 3-6.

Embodiment 18

The method of any one of embodiments 3 to 17, wherein the targetedportion is at most about 1500 nucleotides, about 1000 nucleotides, about800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500nucleotides, about 400 nucleotides, about 300 nucleotides, about 200nucleotides, about 100 nucleotides, about 80 nucleotides, about 70nucleotides, about 60 nucleotides, about 50 nucleotides upstream ofgenomic site GRCh37/hg19: chr2:166,863,803.

Embodiment 19

The method of any one of embodiments 3 to 18, wherein the targetedportion is about 1000 nucleotides, about 800 nucleotides, about 700nucleotides, about 600 nucleotides, about 500 nucleotides, about 400nucleotides, about 300 nucleotides, about 200 nucleotides, about 100nucleotides, about 80 nucleotides, about 70 nucleotides, about 60nucleotides, about 50 nucleotides, about 40 nucleotides, about 30nucleotides, about 20 nucleotides, about 10 nucleotides, about 5nucleotides, about 4 nucleotides, about 2 nucleotides, about 1nucleotides upstream of genomic site GRCh37/hg19: chr2:166,863,803.

Embodiment 20

The method of any one of embodiments 3 to 17, wherein the targetedportion is at most about 1500 nucleotides, about 1000 nucleotides, about800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500nucleotides, about 400 nucleotides, about 300 nucleotides, about 200nucleotides, about 100 nucleotides, about 80 nucleotides, about 70nucleotides, about 60 nucleotides, about 50 nucleotides downstream ofgenomic site GRCh37/hg19: chr2:166,863,740.

Embodiment 21

The method of any one of embodiments 3 to 17 or 20, wherein the targetedportion is about 1000 nucleotides, about 800 nucleotides, about 700nucleotides, about 600 nucleotides, about 500 nucleotides, about 400nucleotides, about 300 nucleotides, about 200 nucleotides, about 100nucleotides, about 80 nucleotides, about 70 nucleotides, about 60nucleotides, about 50 nucleotides, about 40 nucleotides, about 30nucleotides, about 20 nucleotides, about 10 nucleotides, about 5nucleotides, about 4 nucleotides, about 2 nucleotides, about 1nucleotides downstream of genomic site GRCh37/hg19: chr2:166,863,740.

Embodiment 22

The method of any one of embodiments 3 to 21, wherein the targetedportion of the NMD exon mRNA encoding SCN1A comprises a sequence with atleast 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a regioncomprising at least 8 contiguous nucleic acids of SEQ ID NO: SEQ ID NOs:2 or 7-10.

Embodiment 23

The method of embodiment 22, wherein the therapeutic agent is anantisense oligomer (ASO) and wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any oneof SEQ ID NOs: 21-67, 210-256, or 304-379.

Embodiment 24

The method of any one of embodiments 3 to 21, wherein the targetedportion of the NMD exon mRNA encoding SCN1A is within the non-sensemediated RNA decay-inducing exon 20x of SCN1A.

Embodiment 25

The method of embodiment 24, wherein the therapeutic agent is anantisense oligomer (ASO) and wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any oneof SEQ ID NOs: 42-50, or 231-239.

Embodiment 26

The method of any one of embodiments 3 to 21, wherein the targetedportion of the NMD exon mRNA encoding SCN1A is upstream or downstream ofthe non-sense mediated RNA decay-inducing exon 20x of SCN1A.

Embodiment 27

The method of embodiment 26, wherein the therapeutic agent is anantisense oligomer (ASO) and wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any oneof SEQ ID NOs: 21-38, 53-67, 210-227, or 242-256.

Embodiment 28

The method of any one of embodiments 3 to 21, wherein the targetedportion of the NMD exon mRNA comprises an exon-intron junction of exon20x of SCN1A.

Embodiment 29

The method of embodiment 28, wherein the therapeutic agent is anantisense oligomer (ASO) and wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any oneof SEQ ID NOs: 39-41, 51, 52, 228-230, 240, or 241.

Embodiment 30

The method of any one of embodiments 1 to 29, wherein the therapeuticagent promotes exclusion of the NMD exon from the processed mRNAencoding SCN1A protein.

Embodiment 31

The method of embodiment 30, wherein exclusion of the NMD exon from theprocessed mRNA encoding SCN1A protein in the cell contacted with thetherapeutic agent is increased about 1.1 to about 10-fold, about 1.5 toabout 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold,about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 toabout 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 2.5-fold, at leastabout 3-fold, at least about 3.5-fold, at least about 4-fold, at leastabout 5-fold, or at least about 10-fold, compared to exclusion of theNMD exon from the processed mRNA encoding SCN1A protein in a controlcell.

Embodiment 32

The method of embodiment 30 or 31, wherein the therapeutic agentincreases level of the processed mRNA encoding SCN1A protein in thecell.

Embodiment 33

The method of any one of embodiments 30 to 32, wherein an amount of theprocessed mRNA encoding SCN1A protein in the cell contacted with thetherapeutic agent is increased about 1.1 to about 10-fold, about 1.5 toabout 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold,about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 toabout 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 2.5-fold, at leastabout 3-fold, at least about 3.5-fold, at least about 4-fold, at leastabout 5-fold, or at least about 10-fold, compared to an total amount ofthe processed mRNA encoding SCN1A protein in a control cell.

Embodiment 34

The method of any one of embodiments 30 to 33, wherein the therapeuticagent increases expression of SCN1A protein in the cell.

Embodiment 35

The method of any one of embodiments 30 to 34, wherein an amount ofSCN1A produced in the cell contacted with the therapeutic agent isincreased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold,about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 toabout 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold,about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about2-fold, at least about 2.5-fold, at least about 3-fold, at least about3.5-fold, at least about 4-fold, at least about 5-fold, or at leastabout 10-fold, compared to an total amount of SCN1A produced in acontrol cell.

Embodiment 36

The method of any one of embodiments 2 to 35, wherein the disease orcondition is induced by a loss-of-function mutation in Nav1.1.

Embodiment 37

The method of any one of embodiments 2 to 36, wherein the disease orcondition is associated with haploinsufficiency of the SCN1A gene, andwherein the subject has a first allele encoding a functional SCN1A, anda second allele from which SCN1A is not produced or produced at areduced level, or a second allele encoding a nonfunctional SCN1A or apartially functional SCN1A.

Embodiment 38

The method of any one of embodiments 2 to 37, wherein the disease orcondition is encephalopathy.

Embodiment 39

The method of embodiment 38, wherein the encephalopathy is epilepticencephalopathy.

Embodiment 40

The method of any one of embodiments 2 to 37, wherein the disease orcondition is Dravet Syndrome (DS); severe myoclonic epilepsy of infancy(SMEI)-borderland (SMEB); Febrile seizure (FS); epilepsy, generalized,with febrile seizures plus (GEFS+); epileptic encephalopathy, earlyinfantile, 13; cryptogenic generalized epilepsy; cryptogenic focalepilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; Westsyndrome; idiopathic spasms; early myoclonic encephalopathy; progressivemyoclonic epilepsy; alternating hemiplegia of childhood; unclassifiedepileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP);sick sinus syndrome 1; autism; or malignant migrating partial seizuresof infancy.

Embodiment 41

The method of embodiment 40, wherein GEFS+ is epilepsy, generalized,with febrile seizures plus, type 2.

Embodiment 42

The method of embodiment 40, wherein the Febrile seizure is Febrileseizures, familial, 3A.

Embodiment 43

The method of embodiment 40, wherein SMEB is SMEB without generalizedspike wave (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEBlacking more than one feature of SMEI (SMEB-O), or intractable childhoodepilepsy with generalized tonic-clonic seizures (ICEGTC).

Embodiment 44

The method of any one of embodiments 1 to 43, wherein the therapeuticagent promotes exclusion of the NMD exon from the processed mRNAencoding SCN1A protein and increases the expression of SCN1A in thecell.

Embodiment 45

The method of any one of embodiments 1 to 44, wherein the therapeuticagent is an antisense oligomer (ASO) and wherein the ASO comprises asequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100%complimentary to any one of SEQ ID NOs: 22-24, 26, 27, 29-35, 37-62,64-67, or 304-379.

Embodiment 46

The method of any one of embodiments 1 to 29, wherein the therapeuticagent inhibits exclusion of the NMD exon from the processed mRNAencoding SCN1A protein.

Embodiment 47

The method of embodiment 46, wherein exclusion of the NMD exon from theprocessed mRNA encoding SCN1A protein in the cell contacted with thetherapeutic agent is decreased about 1.1 to about 10-fold, about 1.5 toabout 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold,about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 toabout 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 2.5-fold, at leastabout 3-fold, at least about 3.5-fold, at least about 4-fold, at leastabout 5-fold, or at least about 10-fold, compared to exclusion of theNMD exon from the processed mRNA encoding SCN1A protein in a controlcell.

Embodiment 48

The method of embodiment 46 or 47, wherein the therapeutic agentdecreases level of the processed mRNA encoding SCN1A protein in thecell.

Embodiment 49

The method of any one of embodiments 46 to 48, wherein an amount of theprocessed mRNA encoding SCN1A protein in the cell contacted with thetherapeutic agent is decreased about 1.1 to about 10-fold, about 1.5 toabout 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold,about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 toabout 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at leastabout 1.5-fold, at least about 2-fold, at least about 2.5-fold, at leastabout 3-fold, at least about 3.5-fold, at least about 4-fold, at leastabout 5-fold, or at least about 10-fold, compared to an total amount ofthe processed mRNA encoding SCN1A protein in a control cell.

Embodiment 50

The method of any one of embodiments 46 to 49, wherein the therapeuticagent decreases expression of SCN1A protein in the cell.

Embodiment 51

The method of any one of embodiments 46 to 50, wherein an amount ofSCN1A produced in the cell contacted with the therapeutic agent isdecreased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold,about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 toabout 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold,about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about2-fold, at least about 2.5-fold, at least about 3-fold, at least about3.5-fold, at least about 4-fold, at least about 5-fold, or at leastabout 10-fold, compared to an total amount of SCN1A produced in acontrol cell.

Embodiment 52

The method of any one of embodiments 2 to 29 or 46 to 49, wherein thedisease or condition is induced by a gain-of-function mutation inNav1.1.

Embodiment 53

The method of embodiment 52, wherein the subject has an allele fromwhich SCN1A is produced at an increased level, or an allele encoding amutant SCN1A that induces increased activity of Na_(v)1.1 in the cell.

Embodiment 54

The method of embodiment 52 or 53, wherein the disease or condition ismigraine.

Embodiment 55

The method of embodiment 54, wherein the migraine is migraine, familialhemiplegic, 3.

Embodiment 56

The method of any one of embodiments 2 to 49, wherein the disease orcondition is a Na_(v)1.1 genetic epilepsy.

Embodiment 57

The method of any one of embodiments 46 to 56, wherein the therapeuticagent inhibits exclusion of the NMD exon from the processed mRNAencoding SCN1A protein and decreases the expression of SCN1A in thecell.

Embodiment 58

The method of any one of embodiments 46 to 57, wherein the therapeuticagent is an antisense oligomer (ASO) and wherein the ASO comprises asequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100%complimentary to any one of SEQ ID NOs: 21, 25, 28, 36, or 63.

Embodiment 59

The method of any one of previous embodiments, wherein the therapeuticagent is an antisense oligomer (ASO) and wherein the antisense oligomercomprises a backbone modification comprising a phosphorothioate linkageor a phosphorodiamidate linkage.

Embodiment 60

The method of any one of previous embodiments, wherein the therapeuticagent is an antisense oligomer (ASO) and wherein the antisense oligomercomprises a phosphorodiamidate morpholino, a locked nucleic acid, apeptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethylmoiety.

Embodiment 61

The method of any one of previous embodiments, wherein the therapeuticagent is an antisense oligomer (ASO) and wherein the antisense oligomercomprises at least one modified sugar moiety.

Embodiment 62

The method of embodiment 61, wherein each sugar moiety is a modifiedsugar moiety.

Embodiment 63

The method of any one of previous embodiments, wherein the therapeuticagent is an antisense oligomer (ASO) and wherein the antisense oligomerconsists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

Embodiment 64

The method of any one of embodiments 3 to 63, wherein the therapeuticagent is an antisense oligomer (ASO) and wherein the antisense oligomeris at least 80%, at least 85%, at least 90%, at least 95%, at least 98%,at least 99%, or 100%, complementary to the targeted portion of the NMDexon mRNA encoding the protein.

Embodiment 65

The method of any one of previous embodiments, wherein the methodfurther comprises assessing SCN1A mRNA or protein expression.

Embodiment 66

The method of any one of embodiments 2 to 65, wherein the subject is ahuman.

Embodiment 67

The method of any one of embodiments 2 to 65, wherein the subject is anon-human animal.

Embodiment 68

The method of any one of embodiments 2 to 65, wherein the subject is afetus, an embryo, or a child.

Embodiment 69

The method of any one of previous embodiments, wherein the cells are exvivo.

Embodiment 70

The method of any one of embodiments 2 to 69, wherein the therapeuticagent is administered by intrathecal injection, intracerebroventricularinjection, intraperitoneal injection, intramuscular injection,subcutaneous injection, intravitreal, or intravenous injection of thesubject.

Embodiment 71

The method of any one of embodiments 2 to 65, wherein the method furthercomprises administering a second therapeutic agent to the subject.

Embodiment 72

The method of embodiment 71, wherein the second therapeutic agent is asmall molecule.

Embodiment 73

The method of embodiment 71, wherein the second therapeutic agent is anASO.

Embodiment 74

The method of embodiment 73, wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to anyone of SEQ ID NOs: 115-161.

Embodiment 75

The method of embodiment 71, wherein the second therapeutic agentcorrects intron retention.

Embodiment 76

The method of any one of embodiments 2 to 65, wherein the disease orcondition is Alzheimer's Disease, SCN2A encephalopathy, SCN8Aencephalopathy, or SCN5A arrythmia.

Embodiment 77

The method of embodiment 30, 32 or 34, wherein the disease or conditionis Alzheimer's Disease, SCN2A encephalopathy, SCN8A encephalopathy, orSCN5A arrythmia.

Embodiment 78

A method of treating Dravet Syndrome (DS); Epilepsy, generalized, withfebrile seizures plus, type 2; Febrile seizures, familial, 3A; Migraine,familial hemiplegic, 3; Autism; Epileptic encephalopathy, earlyinfantile, 13; Sick sinus syndrome 1; Alzheimer's disease or suddenunexpected death in epilepsy (SUDEP) in a subject in need thereof, byincreasing the expression of a target protein or functional RNA by acell of the subject, wherein the cell has an mRNA that contains anon-sense mediated RNA decay-inducing exon (NMD exon mRNA), and whereinthe NMD exon mRNA encodes the target protein or functional RNA, themethod comprising contacting the cell of the subject with a therapeuticagent that binds to a targeted portion of the NMD exon mRNA encoding thetarget protein or functional RNA, whereby the non-sense mediated RNAdecay-inducing exon is excluded from the NMD exon mRNA encoding thetarget protein or functional RNA, thereby increasing the level ofprocessed mRNA encoding the target protein or functional RNA, andincreasing the expression of the target protein or functional RNA in thecell of the subject.

Embodiment 79

The method of embodiment 78, wherein the target protein is SCN1A.

Embodiment 80

A method of increasing expression of SCN1A protein by a cell having anmRNA that contains a non-sense mediated RNA decay-inducing exon (NMDexon mRNA) and encodes SCN1A protein, the method comprising contactingthe cell an agent that binds to a targeted portion of the NMD exon mRNAencoding SCN1A protein, whereby the non-sense mediated RNAdecay-inducing exon is excluded from the NMD exon mRNA encoding SCN1Aprotein, thereby increasing the level of processed mRNA encoding SCN1Aprotein, and increasing the expression of SCN1A protein in the cell.

Embodiment 81

A method of treating a disease or condition in a subject in need thereofby increasing the expression of SCN1A protein in a cell of the subject,comprising: contacting the cell of the subject with a therapeutic agentthat binds to a targeted portion of a non-sense mediated RNAdecay-inducing exon mRNA encoding the SCN1A protein or functional SCN1ARNA, whereby the non-sense mediated RNA decay-inducing exon is excludedfrom the NMD exon mRNA encoding the SCN1A protein or functional SCN1ARNA, thereby increasing the level of processed mRNA encoding the SCN1Aprotein or functional SCN1A RNA, and increasing the expression of theSCN1A protein or functional SCN1A RNA in the cell of the subject;wherein the disease or condition is associated with a mutation of a geneother than an SCN1A gene, aberrant expression of a protein encoded by agene other than an SCN1A gene or aberrant expression of an RNA encodedby a gene other than an SCN1A gene.

Embodiment 82

The method of embodiment 81, wherein a symptom of the disease orcondition is reduced by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more.

Embodiment 83

The method of embodiment 81 or 82, wherein a symptom of the disease orcondition is reduced by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or 100% with an increase in expression of the SCN1A protein.

Embodiment 84

The method of any one of embodiments 81 to 83, wherein progression ofthe disease or condition is reduced by about 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more with anincrease in expression of the SCN1A protein.

Embodiment 85

The method of any one of embodiments 81 to 84, wherein progression ofthe disease or condition is reduced by about 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or 100% with an increase in expression of the SCN1Aprotein.

Embodiment 86

The method of any one of embodiments 81 to 85, wherein increasing theexpression of the SCN1A protein or functional SCN1A RNA compensates forthe mutation of a gene other than an SCN1A gene, the aberrant expressionof a protein encoded by a gene other than an SCN1A gene or the aberrantexpression of an RNA encoded by a gene other than an SCN1A gene.

Embodiment 87

The method of any one of embodiments 81 to 86, wherein the disease orcondition is epileptic encephalopathy, early infantile, 13.

Embodiment 88

The method of any one of embodiments 81 to 87, wherein the subject has amutation in the SCN8A gene.

Embodiment 89

The method of any one of embodiments 81 to 86, wherein the disease orcondition is sick sinus syndrome 1.

Embodiment 90

The method of any one of embodiments 81 to 86 or 88, wherein the subjecthas a mutation in the SCN5A gene

Embodiment 91

The method of any one of embodiments 81 to 86, wherein the disease orcondition is Alzheimer's disease.

Embodiment 92

A method of treating a disease or condition in a subject in needthereof, comprising administering to the subject a compositioncomprising an antisense oligomer, the antisense oligomer comprising asequence of at least 8 contiguous nucleotides that is at least 80%, 85%,90%, 95%, 97%, or 100% complementary to intron 20 of SCN1A.

Embodiment 93

A method of treating a disease or condition in a subject in needthereof, comprising administering to the subject a compositioncomprising an antisense oligomer, the antisense oligomer comprising asequence of at least 8 contiguous nucleotides that is at least 80%, 85%,90%, 95%, 97%, or 100% complementary to any one of SEQ ID NOs: 7-10.

Embodiment 94

The method of any one of embodiments 78 to 93, wherein the non-sensemediated RNA decay-inducing exon is spliced out from the NMD exon mRNAencoding the target protein or functional RNA.

Embodiment 95

The method of any one of embodiments 78 to 94, wherein the targetprotein does not comprise an amino acid sequence encoded by thenon-sense mediated RNA decay-inducing exon.

Embodiment 96

The method of any one of embodiments 78 to 95, wherein the targetprotein is a full-length target protein.

Embodiment 97

The method of any one of embodiments 78 to 96, wherein the agent is anantisense oligomer (ASO) complementary to the targeted portion of theNMD exon mRNA.

Embodiment 98

The method of any one of embodiments 78 to 97, wherein the mRNA ispre-mRNA.

Embodiment 99

The method of any one of embodiments 78 to 98, wherein the contactingcomprises contacting the therapeutic agent to the mRNA, wherein the mRNAis in a nucleus of the cell.

Embodiment 100

The method of any one of embodiments 78 to 99, wherein the targetprotein or the functional RNA corrects a deficiency in the targetprotein or functional RNA in the subject.

Embodiment 101

The method of any one of embodiments 78 to 100, wherein the cells are inor from a subject with a condition caused by a deficient amount oractivity of SCN1A protein.

Embodiment 102

The method of any one of embodiments 78 to 101, wherein the deficientamount of the target protein is caused by haploinsufficiency of thetarget protein, wherein the subject has a first allele encoding afunctional target protein, and a second allele from which the targetprotein is not produced or produced at a reduced level, or a secondallele encoding a nonfunctional or partially functional target protein,and wherein the antisense oligomer binds to a targeted portion of a NMDexon mRNA transcribed from the first allele.

Embodiment 103

The method of any one of embodiments 78 to 101, wherein the subject hasa condition caused by a disorder resulting from a deficiency in theamount or function of the target protein, wherein the subject has

(a) a first mutant allele from which

-   -   (i) the target protein is produced at a reduced level compared        to production from a wild-type allele,    -   (ii) the target protein is produced in a form having reduced        function compared to an equivalent wild-type protein, or    -   (iii) the target protein is not produced, and        (b) a second mutant allele from which    -   (i) the target protein is produced at a reduced level compared        to production from a wild-type allele,    -   (ii) the target protein is produced in a form having reduced        function compared to an equivalent wild-type protein, or    -   (iii) the target protein is not produced, and    -   wherein when the subject has a first mutant allele (a)(iii), the        second mutant allele is (b)(i) or (b)(ii) and wherein when the        subject has a second mutant allele (b)(iii), the first mutant        allele is (a)(i) or (a)(ii), and wherein the NMD exon mRNA is        transcribed from either the first mutant allele that is (a)(i)        or (a)(ii), and/or the second allele that is (b)(i) or (b)(ii).

Embodiment 104

The method of embodiment 103, wherein the target protein is produced ina form having reduced function compared to the equivalent wild-typeprotein.

Embodiment 105

The method of embodiment 103, wherein the target protein is produced ina form that is fully-functional compared to the equivalent wild-typeprotein.

Embodiment 106

The method of any one of embodiments 78 to 105, wherein the targetedportion of the NMD exon mRNA is within the non-sense mediated RNAdecay-inducing exon.

Embodiment 107

The method of any one of embodiments 78 to 105, wherein the targetedportion of the NMD exon mRNA is either upstream or downstream of thenon-sense mediated RNA decay-inducing exon.

Embodiment 108

The method of any one of embodiments 78 to 107, wherein the NMD exonmRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%,or 100% sequence identity to any one of SEQ ID NOs: 2, 7-10, 12, and17-20.

Embodiment 109

The method of any one of embodiments 78 to 107, wherein the NMD exonmRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%,95%, 97%, or 100% sequence identity to SEQ ID NOs: 1, 3-6, 11, and13-16.

Embodiment 110

The method of any one of embodiments 78 to 107, wherein the targetedportion of the NMD exon mRNA comprises a sequence with at least 80%,85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising atleast 8 contiguous nucleic acids of SEQ ID NO: SEQ ID NOs: 2, 7-10, 12,and 17-20.

Embodiment 111

The method of any one of embodiments 78 to 110, wherein the agent is anantisense oligomer (ASO) and wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any oneof SEQ ID NOs: 21-114.

Embodiment 112

The method of any one of embodiments 78 to 105, wherein the targetedportion of the NMD exon mRNA is within the non-sense mediated RNAdecay-inducing exon 20x of SCN1A.

Embodiment 113

The method of embodiment 112, wherein the agent is an antisense oligomer(ASO) and wherein the ASO comprises a sequence that is at least about80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs:42-50, or 231-239.

Embodiment 114

The method of embodiment any one of embodiments 78 to 105, wherein thetargeted portion of the NMD exon mRNA is upstream or downstream of thenon-sense mediated RNA decay-inducing exon 20x of SCN1A.

Embodiment 115

The method of embodiment 114, wherein the agent is an antisense oligomer(ASO) and wherein the ASO comprises a sequence that is at least about80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs:21-38, 53-67, 210-227, or 242-256.

Embodiment 116

The method of any one of embodiments 78 to 105, wherein the targetedportion of the NMD exon mRNA comprises an exon-intron junction of exon20x of SCN1A.

Embodiment 117

The method of embodiment 116, wherein the agent is an antisense oligomer(ASO) and wherein the ASO comprises a sequence that is at least about80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs:39-41, 51, 52, 228-230, 240, or 241.

Embodiment 118

The method of any one of embodiments 78 to 105, wherein the targetedportion of the NMD exon mRNA is within the non-sense mediated RNAdecay-inducing exon 21x of Scn1a.

Embodiment 119

The method of embodiment 118, wherein the agent is an antisense oligomer(ASO) and wherein the ASO comprises a sequence that is at least about80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs:89-97.

Embodiment 120

The method of embodiment any one of embodiments 78 to 105, wherein thetargeted portion of the NMD exon mRNA is either upstream or downstreamof the non-sense mediated RNA decay-inducing exon 21x of Scn1a.

Embodiment 121

The method of embodiment 120, wherein the agent is an antisense oligomer(ASO) and wherein the ASO comprises a sequence that is at least about80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs:68-85 and 100-114.

Embodiment 122

The method of any one of embodiments 78 to 105, wherein the targetedportion of the NMD exon mRNA comprises an exon-intron junction of exon21x of Scn1a.

Embodiment 123

The method of embodiment 122, wherein the agent is an antisense oligomer(ASO) and wherein the ASO comprises a sequence that is at least about80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs:86-88 and 98-99.

Embodiment 124

The method of any one of embodiments 78 to 123, wherein the targetprotein produced is full-length protein, or wild-type protein.

Embodiment 125

The method of any one of embodiments 78 to 124, wherein the total amountof the processed mRNA encoding the target protein or functional RNAproduced in the cell contacted with the antisense oligomer is increasedabout 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold,about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 toabout 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, atleast about 1.1-fold, at least about 1.5-fold, at least about 2-fold, atleast about 2.5-fold, at least about 3-fold, at least about 3.5-fold, atleast about 4-fold, at least about 5-fold, or at least about 10-fold,compared to the total amount of the processed mRNA encoding the targetprotein or functional RNA produced in a control cell.

Embodiment 126

The method of one any of embodiments 78 to 124, wherein the total amountof target protein produced by the cell contacted with the antisenseoligomer is increased about 1.1 to about 10-fold, about 1.5 to about10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 toabout 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold,about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 toabout 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold,about 4 to about 9-fold, at least about 1.1-fold, at least about1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about3-fold, at least about 3.5-fold, at least about 4-fold, at least about5-fold, or at least about 10-fold, compared to the total amount oftarget protein produced by a control cell.

Embodiment 127

The method of any one of embodiments 78 to 126, wherein the agent is anantisense oligomer (ASO) and wherein the antisense oligomer comprises abackbone modification comprising a phosphorothioate linkage or aphosphorodiamidate linkage.

Embodiment 128

The method of any one of embodiments 78 to 127, wherein the agent is anantisense oligomer (ASO) and wherein the antisense oligomer comprises aphosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleicacid, a 2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety.

Embodiment 129

The method of any one of embodiments 78 to 128, wherein the agent is anantisense oligomer (ASO) and wherein the antisense oligomer comprises atleast one modified sugar moiety.

Embodiment 130

The method of embodiment 129, wherein each sugar moiety is a modifiedsugar moiety.

Embodiment 131

The method of any one of embodiments 78 to 130, wherein the agent is anantisense oligomer (ASO) and wherein the antisense oligomer consists offrom 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20nucleobases, or 12 to 15 nucleobases.

Embodiment 132

The method of any one of embodiments 78 to 131, wherein the agent is anantisense oligomer (ASO) and wherein the antisense oligomer is at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%, complementary to the targeted portion of the NMD exon mRNAencoding the protein.

Embodiment 133

The method of any one of embodiments 78 to 132, wherein the methodfurther comprises assessing SCN1A mRNA or protein expression.

Embodiment 134

The method of any one of embodiments 1 to 133, wherein Dravet Syndrome;Epilepsy, generalized, with febrile seizures plus, type 2; Febrileseizures, familial, 3A; Migraine, familial hemiplegic, 3; Autism;Epileptic encephalopathy, early infantile, 13; Sick sinus syndrome 1;Alzheimer's disease or sudden unexpected death in epilepsy (SUDEP) istreated and wherein the antisense oligomer binds to a targeted portionof a SCN1A NMD exon mRNA, wherein the targeted portion is within asequence selected from SEQ ID NOs: 7-10 and 17-20.

Embodiment 135

The method of any one of embodiments 78 to 134, wherein the subject is ahuman.

Embodiment 136

The method of any one of embodiments 78 to 135, wherein the subject is anon-human animal.

Embodiment 137

The method of any one of embodiments 78 to 136, wherein the subject is afetus, an embryo, or a child.

Embodiment 138

The method of any one of embodiments 78 to 137, wherein the cells are exvivo.

Embodiment 139

The method of any one of embodiments 78 to 138, wherein the therapeuticagent is administered by intrathecal injection, intracerebroventricularinjection, intraperitoneal injection, intramuscular injection,subcutaneous injection, intravitreal injection, or intravenous injectionof the subject.

Embodiment 140

The method of any of embodiments 78 to 139, wherein the method furthercomprises administering a second therapeutic agent to the subject.

Embodiment 141

The method of embodiment 140, wherein the second therapeutic agent is asmall molecule.

Embodiment 142

The method of embodiment 140, wherein the second therapeutic agent is anASO.

Embodiment 143

The method of embodiment 142, wherein the ASO comprises a sequence thatis at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any oneof SEQ ID NOs: 115-161.

Embodiment 144

The method of any one of embodiments 140 to 142, wherein the secondtherapeutic agent corrects intron retention.

Embodiment 145

An antisense oligomer as used in a method of any of embodiments 78 to144.

Embodiment 146

An antisense oligomer comprising a sequence with at least about 80%,85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs:21-114.

Embodiment 147

A pharmaceutical composition comprising the antisense oligomer ofembodiment 145 or 146 and an excipient.

Embodiment 148

A method of treating a subject in need thereof, comprising administeringthe pharmaceutical composition of embodiment 147 to the subject, whereinthe administering is by intrathecal injection, intracerebroventricularinjection, intraperitoneal injection, intramuscular injection,subcutaneous injection, intravitreal injection, or intravenousinjection.

Embodiment 149

A composition comprising a therapeutic agent for use in a method ofincreasing expression of a target protein or a functional RNA by cellsto treat a disease or condition associated with a deficient protein ordeficient functional RNA in a subject in need thereof, wherein thedeficient protein or deficient functional RNA is deficient in amount oractivity in the subject, wherein the target protein is:

(a) the deficient protein; or

(b) a compensating protein which functionally augments or replaces thedeficient protein or in the subject;

and wherein the functional RNA is:

(c) the deficient RNA; or

(d) a compensating functional RNA which functionally augments orreplaces the deficient functional RNA in the subject;

wherein the therapeutic agent enhances exclusion of the non-sensemediated RNA decay-inducing exon from the NMD exon mRNA encoding thetarget protein or functional RNA, thereby increasing production oractivity of the target protein or the functional RNA in the subject.

Embodiment 150

A composition comprising a therapeutic agent for use in a method oftreating a disease or condition in a subject in need thereof, the methodcomprising the step of modulating expression of SCN1A protein by cellsof the subject, wherein the cells have an mRNA that contains a non-sensemediated RNA decay-inducing exon (NMD exon mRNA) and encodes SCN1Aprotein, the method comprising contacting the cells with the therapeuticagent, whereby exclusion of the non-sense mediated RNA decay-inducingexon from the NMD exon mRNA that encodes SCN1A protein is modulated,thereby modulating the level of processed mRNA encoding SCN1A protein,and modulating the expression of SCN1A protein in the cells of thesubject.

Embodiment 151

The composition of embodiment 150, wherein the disease or condition isselected from the group consisting of: Dravet Syndrome (DS); severemyoclonic epilepsy of infancy (SMEI)-borderland (SMEB); Febrile seizure(FS); epilepsy, generalized, with febrile seizures plus (GEFS+);epileptic encephalopathy, early infantile, 13; cryptogenic generalizedepilepsy; cryptogenic focal epilepsy; myoclonic-astatic epilepsy;Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; earlymyoclonic encephalopathy; progressive myoclonic epilepsy; alternatinghemiplegia of childhood; unclassified epileptic encephalopathy; suddenunexpected death in epilepsy (SUDEP); sick sinus syndrome 1; autism; ormigraine, familial hemiplegic, 3; and Alzheimer's Diseases.

Embodiment 152

The composition of any one of embodiments 150 to 151, wherein the SCN1Aprotein and NMD exon mRNA are encoded by the SCN1A gene.

Embodiment 153

The composition of any one of embodiments 149 to 152, wherein thenon-sense mediated RNA decay-inducing exon is spliced out from the NMDexon mRNA encoding the SCN1A protein.

Embodiment 154

The composition of any one of embodiments 149 to 153, wherein the SCN1Aprotein does not comprise an amino acid sequence encoded by thenon-sense mediated RNA decay-inducing exon.

Embodiment 155

The composition of any one of embodiments 149 to 154, wherein the SCN1Aprotein is a full-length SCN1A protein.

Embodiment 156

The composition of any one of embodiments 149 to 155, wherein thetherapeutic agent is an antisense oligomer (ASO) complementary to thetargeted portion of the NMD exon mRNA.

Embodiment 157

The composition of any of embodiments 149 to 156, wherein thetherapeutic agent is an antisense oligomer (ASO) and wherein theantisense oligomer targets a portion of the NMD exon mRNA that is withinthe non-sense mediated RNA decay-inducing exon.

Embodiment 158

The composition of any of embodiments 149 to 156, wherein thetherapeutic agent is an antisense oligomer (ASO) and wherein theantisense oligomer targets a portion of the NMD exon mRNA that isupstream or downstream of the non-sense mediated RNA decay-inducingexon.

Embodiment 159

The composition of any one of embodiments 149 to 158, wherein the targetprotein is SCN1A.

Embodiment 160

The composition of embodiment 159, wherein the NMD exon mRNA comprises asequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequenceidentity to any one of SEQ ID NOs: 2, 7-10, 12, and 17-20.

Embodiment 161

The composition of embodiment 159, wherein the NMD exon mRNA is encodedby a genetic sequence with at least about 80%, 85%, 90%, 95%, 97%, or100% sequence identity to SEQ ID NO: 1, 3-6, 11, and 13-16.

Embodiment 162

The composition of embodiment 159, wherein the targeted portion of theNMD exon mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,97%, or 100% sequence identity to a region comprising at least 8contiguous nucleic acids of SEQ ID NO: 2, 7-10, 12, and 17-20.

Embodiment 163

The composition of any one of embodiments 159 to 162, wherein thetargeted portion of the NMD exon mRNA (i) is within non-sense mediatedRNA decay-inducing exon 20x, (ii) is upstream or downstream of non-sensemediated RNA decay-inducing exon 20x, or (iii) comprises an exon-intronjunction of non-sense mediated RNA decay-inducing exon 20x.

Embodiment 164

The composition of any one of embodiments 159 to 163, wherein thetherapeutic agent is an antisense oligomer (ASO) and wherein the ASOcomprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or100% identity to any one of SEQ ID NOs: 21-114.

Embodiment 165

The composition of any one of embodiments 149 to 164, wherein thedisease or condition is induced by a loss-of-function mutation inNav1.1.

Embodiment 166

The composition of any one of embodiments 149 to 165, wherein thedisease or condition is associated with haploinsufficiency of the SCN1Agene, and wherein the subject has a first allele encoding a functionalSCN1A, and a second allele from which SCN1A is not produced or producedat a reduced level, or a second allele encoding a nonfunctional SCN1A ora partially functional SCN1A.

Embodiment 167

The composition of any one of embodiments 149 to 166, wherein thedisease or condition is encephalopathy, optionally induced by aloss-of-function mutation in Nav1.1.

Embodiment 168

The composition of embodiment 167, wherein the encephalopathy isepileptic encephalopathy.

Embodiment 169

The composition of embodiment 165 or 166, wherein the disease orcondition is Dravet Syndrome (DS); severe myoclonic epilepsy of infancy(SMEI)-borderland (SMEB); Febrile seizure (FS); epilepsy, generalized,with febrile seizures plus (GEFS+); epileptic encephalopathy, earlyinfantile, 13; cryptogenic generalized epilepsy; cryptogenic focalepilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; Westsyndrome; idiopathic spasms; early myoclonic encephalopathy; progressivemyoclonic epilepsy; alternating hemiplegia of childhood; unclassifiedepileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP);sick sinus syndrome 1; autism; or malignant migrating partial seizuresof infancy.

Embodiment 170

The composition of embodiment 168, wherein GEFS+ is epilepsy,generalized, with febrile seizures plus, type 2.

Embodiment 171

The composition of embodiment 168, wherein the Febrile seizure isFebrile seizures, familial, 3A.

Embodiment 172

The composition of embodiment 168, wherein SMEB is SMEB withoutgeneralized spike wave (SMEB-SW), SMEB without myoclonic seizures(SMEB-M), SMEB lacking more than one feature of SMEI (SMEB-O), orintractable childhood epilepsy with generalized tonic-clonic seizures(ICEGTC).

Embodiment 173

The composition of any one of embodiments 165 to 172, wherein thetherapeutic agent promotes exclusion of the NMD exon from the processedmRNA encoding SCN1A protein and increases the expression of SCN1A in thecell.

Embodiment 174

The composition of any one of embodiments 165 to 173, wherein thetherapeutic agent is an antisense oligomer (ASO) and wherein the ASOcomprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or100% complimentary to any one of SEQ ID NOs: 22-24, 26, 27, 29-35,37-62, or 64-67.

Embodiment 175

The composition of any one of embodiments 149 to 164, wherein thedisease or condition is induced by a gain-of-function mutation inNav1.1.

Embodiment 176

The composition of any one of embodiments 149 to 164 or 175, wherein thesubject has an allele from which SCN1A is produced at an increasedlevel, or an allele encoding a mutant SCN1A that induces increasedactivity of Na_(v)1.1 in the cell.

Embodiment 177

The composition of any one of embodiments 149 to 164, 175, or 176,wherein the disease or condition is migraine.

Embodiment 178

The composition of embodiment 177, wherein the migraine is migraine,familial hemiplegic, 3.

Embodiment 179

The composition of any one of embodiments 149 to 164, 175, or 176,wherein the disease or condition is a Na_(v)1.1 genetic epilepsy.

Embodiment 180

The composition of any one of embodiments 149 to 164, or 175 to 179,wherein the therapeutic agent inhibits exclusion of the NMD exon fromthe processed mRNA encoding SCN1A protein and decreases the expressionof SCN1A in the cell.

Embodiment 181

The composition of any one of embodiments 149 to 164, or 175 to 180,wherein the therapeutic agent is an antisense oligomer (ASO) and whereinthe ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%,97%, or 100% complimentary to any one of SEQ ID NOs: 21, 25, 28, 36, or63.

Embodiment 182

The composition of any one of embodiments 149 to 181, wherein theprocessed mRNA encoding the target protein or functional RNA is afull-length mature mRNA, or a wild-type mature mRNA.

Embodiment 183

The composition of any one of embodiments 149 to 182, wherein the targetprotein produced is full-length protein, or wild-type protein.

Embodiment 184

The composition of any one of embodiments 149 to 183, wherein thetherapeutic agent is an antisense oligomer (ASO) and wherein theantisense oligomer comprises a backbone modification comprising aphosphorothioate linkage or a phosphorodiamidate linkage.

Embodiment 185

The composition of any of embodiments 149 to 184 wherein the therapeuticagent is an antisense oligomer (ASO) and wherein said antisense oligomeris an antisense oligonucleotide.

Embodiment 186

The composition of any of embodiments 149 to 185, wherein thetherapeutic agent is an antisense oligomer (ASO) and wherein theantisense oligomer comprises a phosphorodiamidate morpholino, a lockednucleic acid, a peptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a2′-O-methoxyethyl moiety.

Embodiment 187

The composition of any of embodiments 149 to 186, wherein thetherapeutic agent is an antisense oligomer (ASO) and wherein theantisense oligomer comprises at least one modified sugar moiety.

Embodiment 188

The composition of embodiment 187, wherein each sugar moiety is amodified sugar moiety.

Embodiment 189

The composition of any of embodiments 149 to 188, wherein thetherapeutic agent is an antisense oligomer (ASO) and wherein theantisense oligomer consists of from 8 to 50 nucleobases, 8 to 40nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15nucleobases.

Embodiment 190

A composition comprising an antisense oligomer, the antisense oligomercomprising a sequence of at least 8 contiguous nucleotides that is atleast 80%, 85%, 90%, 95%, 97%, or 100% complementary to intron 20 ofSCN1A.

Embodiment 191

A composition comprising an antisense oligomer, the antisense oligomercomprising a sequence of at least 8 contiguous nucleotides that is atleast 80%, 85%, 90%, 95%, 97%, or 100% complementary to any one of SEQID NOs: 7-10.

Embodiment 192

A pharmaceutical composition comprising the therapeutic agent of any ofthe compositions of embodiments 149 to 191, and an excipient.

Embodiment 193

A method of treating a subject in need thereof, comprising administeringthe pharmaceutical composition of embodiment 192 to the subject, whereinthe administering is by intrathecal injection, intracerebroventricularinjection, intraperitoneal injection, intramuscular injection,subcutaneous injection, intravitreal injection, or intravenousinjection.

Embodiment 194

A pharmaceutical composition comprising: an antisense oligomer thathybridizes to a target sequence of a SCN1A mRNA transcript, wherein theSCN1A mRNA transcript comprises a non-sense mediated RNA decay-inducingexon, wherein the antisense oligomer induces exclusion of the non-sensemediated RNA decay-inducing exon from the SCN1A mRNA transcript; and apharmaceutical acceptable excipient.

Embodiment 195

The pharmaceutical composition of embodiment 194, wherein the SCN1A mRNAtranscript is a SCN1A NMD exon mRNA transcript.

Embodiment 196

The pharmaceutical composition of embodiment 194 or 195, wherein thetargeted portion of the SCN1A NMD exon mRNA transcript (i) is withinnon-sense mediated RNA decay-inducing exon 20x, (ii) is upstream ordownstream of non-sense mediated RNA decay-inducing exon 20x, or (iii)comprises an exon-intron junction of non-sense mediated RNAdecay-inducing exon 20x.

Embodiment 197

The pharmaceutical composition of embodiment 194 or 196, wherein theSCN1A NMD exon mRNA transcript is encoded by a genetic sequence with atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to any one of SEQ ID NOs: 1, 3-6, 11, and 13-16.

Embodiment 198

The pharmaceutical composition of embodiment 194 or 196, wherein theSCN1A NMD exon mRNA transcript comprises a sequence with at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to anyone of SEQ ID NOs: 2, 7-10, 12, and 17-20.

Embodiment 199

The pharmaceutical composition of embodiment 194, wherein the antisenseoligomer comprises a backbone modification comprising a phosphorothioatelinkage or a phosphorodiamidate linkage.

Embodiment 200

The pharmaceutical composition of embodiment 194, wherein the antisenseoligomer is an antisense oligonucleotide.

Embodiment 201

The pharmaceutical composition of embodiment 194, wherein the antisenseoligomer comprises a phosphorodiamidate morpholino, a locked nucleicacid, a peptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a2′-O-methoxyethyl moiety.

Embodiment 202

The pharmaceutical composition of embodiment 194, wherein the antisenseoligomer comprises at least one modified sugar moiety.

Embodiment 203

The pharmaceutical composition of embodiment 194, wherein the antisenseoligomer comprises from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

Embodiment 204

The pharmaceutical composition of embodiment 194 or 195, wherein theantisense oligomer is at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99%, or is 100% complementary to a targetedportion of the SCN1A NMD exon mRNA transcript.

Embodiment 205

The pharmaceutical composition of embodiment 194 or 195 wherein thetargeted portion of the SCN1A NMD exon mRNA transcript is within asequence selected from SEQ ID NOs: 2, 7-10, 12, and 17-20.

Embodiment 206

The pharmaceutical composition of embodiment 194, wherein the antisenseoligomer comprises a nucleotide sequence that is at least about 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any one of SEQ ID NOs: 21-114.

Embodiment 207

The pharmaceutical composition of embodiment 194, wherein the antisenseoligomer comprises a nucleotide sequence selected from SEQ ID NOs:21-114.

Embodiment 208

The pharmaceutical composition of any one of the embodiments 194 to 207,wherein the pharmaceutical composition is formulated for intrathecalinjection, intracerebroventricular injection, intraperitoneal injection,intramuscular injection, subcutaneous injection, intravitreal injection,or intravenous injection.

Embodiment 209

A method of inducing processing of a deficient SCN1A mRNA transcript tofacilitate removal of a non-sense mediated RNA decay-inducing exon toproduce a fully processed SCN1A mRNA transcript that encodes afunctional form of a SCN1A protein, the method comprising:

(a) contacting an antisense oligomer to a target cell of a subject;

(b) hybridizing the antisense oligomer to the deficient SCN1A mRNAtranscript, wherein the deficient SCN1A mRNA transcript is capable ofencoding the functional form of a SCN1A protein and comprises at leastone non-sense mediated RNA decay-inducing exon;

(c) removing the at least one non-sense mediated RNA decay-inducing exonfrom the deficient SCN1A mRNA transcript to produce the fully processedSCN1A mRNA transcript that encodes the functional form of SCN1A protein;and

(d) translating the functional form of SCN1A protein from the fullyprocessed SCN1A mRNA transcript.

Embodiment 210

A method of treating a subject having a condition caused by a deficientamount or activity of SCN1A protein comprising administering to thesubject an antisense oligomer comprising a nucleotide sequence with atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to any one of SEQ ID NOs: 24-114.

Embodiment 211

A method of screening for an agent that increases gene expression of atarget protein or functional RNA by a cell, wherein the cell has an mRNAthat contains a non-sense mediated RNA decay-inducing exon (NMD exonmRNA), and wherein the NMD exon mRNA encodes the target protein orfunctional RNA, the method comprising

(a) contacting a test agent that targets the NMD exon mRNA to a firstcell;

(b) contacting a control agent to a second cell;

(c) determining a first level in the first cell, wherein the first levelis a level of (i) an RNA transcript encoded by the NMD exon mRNA thatdoes not comprise the RNA decay-inducing exon, or (ii) a protein encodedby the NMD exon mRNA that does not comprise an amino acid sequenceencoded by the RNA decay-inducing exon;(d) determining a second level in the second cell, wherein the secondlevel is a level of (i) an RNA transcript encoded by the NMD exon mRNAthat does not comprise the RNA decay-inducing exon, or (ii) a proteinencoded by the NMD exon mRNA that does not comprise an amino acidsequence encoded by the RNA decay-inducing exon;wherein the first level is higher than the second level; and(e) selecting the test agent.

Embodiment 212

A method of screening for an agent that increases gene expression of atarget protein or functional RNA by a cell, wherein the cell has an mRNAthat contains a non-sense mediated RNA decay-inducing exon (NMD exonmRNA), and wherein the NMD exon mRNA encodes the target protein orfunctional RNA, the method comprising

(a) contacting a test agent that targets the NMD exon mRNA to a firstcell;

(b) contacting a control agent to a second cell;

(c) determining a first level in the first cell, wherein the first levelis a level of (i) an RNA transcript encoded by the NMD exon mRNA thatcomprises the RNA decay-inducing exon, or (ii) a protein encoded by theNMD exon mRNA that comprises an amino acid sequence encoded by the RNAdecay-inducing exon;(d) determining a second level in the second cell, wherein the secondlevel is a level of (i) an RNA transcript encoded by the NMD exon mRNAthat comprises the RNA decay-inducing exon, or (ii) a protein encoded bythe NMD exon mRNA that comprises an amino acid sequence encoded by theRNA decay-inducing exon;wherein the first level is lower than the second level; and(e) selecting the test agent.

Embodiment 213

The method of embodiment 211 or 212, wherein the method comprisescontacting a protein synthesis inhibitor to the first cell and thesecond cell; wherein the first level is a level of an RNA transcriptencoded by the NMD exon mRNA that comprises the RNA decay-inducing exon;and wherein the second level is a level of an RNA transcript encoded bythe NMD exon mRNA that comprises the RNA decay-inducing exon.

Embodiment 214

A method of treating Dravet Syndrome (DS), Epilepsy, generalized, withfebrile seizures plus, type 2; Febrile seizures, familial, 3A; Migraine,familial hemiplegic, 3; Autism; Epileptic encephalopathy, earlyinfantile, 13; Sick sinus syndrome 1; Alzheimer's disease or SUDEP(sudden unexpected death in epilepsy) in a subject in need thereof, byincreasing the expression of a target protein or functional RNA by acell of the subject, wherein the cell has an mRNA that contains anon-sense mediated RNA decay-inducing exon (NMD exon mRNA), and whereinthe NMD exon mRNA encodes the target protein or functional RNA, themethod comprising contacting the cell of the subject with a therapeuticagent that modulates splicing of the NMD exon mRNA encoding the targetprotein or functional RNA, whereby the non-sense mediated RNAdecay-inducing exon is excluded from the NMD exon mRNA encoding thetarget protein or functional RNA, thereby increasing the level ofprocessed mRNA encoding the target protein or functional RNA, andincreasing the expression of the target protein or functional RNA in thecell of the subject.

Embodiment 215

A method of increasing expression of SCN1A protein by a cell having anmRNA that contains a non-sense mediated RNA decay-inducing exon (NMDexon mRNA) and encodes SCN1A protein, the method comprising contactingthe cell an agent that modulates splicing of the NMD exon mRNA encodingSCN1A protein, whereby the non-sense mediated RNA decay-inducing exon isexcluded from the NMD exon mRNA encoding SCN1A protein, therebyincreasing the level of processed mRNA encoding SCN1A protein, andincreasing the expression of SCN1A protein in the cell.

Embodiment 216

The method of embodiment 214 or 215, wherein the agent

(a) binds to a targeted portion of the NMD exon mRNA encoding the targetprotein or functional RNA;

(b) binds to one or more components of a spliceosome; or

(c) a combination of (a) and (b).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES

The present invention will be more specifically illustrated by thefollowing Examples. However, it should be understood that the presentinvention is not limited by these examples in any manner.

Example 1: Identification of NMD-Inducing Exon Inclusion Events in SCN1ATranscripts by RNAseq Using Next Generation Sequencing

Whole transcriptome shotgun sequencing was carried out using nextgeneration sequencing to reveal a snapshot of transcripts produced bythe SCN1A gene to identify NIE inclusion events. For this purpose,polyA+RNA from nuclear and cytoplasmic fractions of HCN (human corticalneurons) was isolated and cDNA libraries constructed using Illumina'sTruSeq Stranded mRNA library Prep Kit. The libraries were pair-endsequenced resulting in 100-nucleotide reads that were mapped to thehuman genome (February 2009, GRCh37/hg19 assembly). The sequencingresults for SCN1A are shown in FIG. 2. Briefly, FIG. 2 shows the mappedreads visualized using the UCSC genome browser (operated by the UCSCGenome Informatics Group (Center for Biomolecular Science & Engineering,University of California, Santa Cruz, 1156 High Street, Santa Cruz,Calif. 95064) and described by, e.g., Rosenbloom, et al., 2015, “TheUCSC Genome Browser database: 2015 update,” Nucleic Acids Research 43,Database Issue, doi: 10.1093/nar/gku1177) and the coverage and number ofreads can be inferred by the peak signals. The height of the peaksindicates the level of expression given by the density of the reads in aparticular region. The upper panel shows a graphic representation of theSCN1A gene to scale. The conservation level across 100 vertebratespecies is shown as peaks. The highest peaks correspond to exons (blackboxes), while no peaks are observed for the majority of the introns(lines with arrow heads). Peaks of conservation were identified inintron 20 (NM_006920), shown in the middle panel. Inspection of theconserved sequences identified an exon-like sequence of 64 bp (bottompanel, sequence highlighted in grey) flanked by 3′ and 5′ splice sites(underlined sequence). Inclusion of this exon leads to a frameshift andthe introduction of a premature termination codon in exon 21 renderingthe transcript a target of NMD.

Exemplary SCN1A gene, pre-mRNA, exon, and intron sequences aresummarized in Table 2. The sequence for each exon or intron issummarized in Table 3.

TABLE 2 List of target SCN1A gene and pre-mRNA sequences. Species SEQ IDNO. Sequence Type Human SEQ ID NO. 1 SCN1A gene (NC_000002.12) SEQ IDNO. 2 SCN1A pre-mRNA (encoding e.g., SCN1A mRNA NM_006920.5) SEQ ID NO.3 Exon 20 gene SEQ ID NO. 4 Intron 20 gene SEQ ID NO. 5 Exon 21 gene SEQID NO. 6 Exon 20x gene SEQ ID NO. 7 Exon 20 pre-mRNA SEQ ID NO. 8 Intron20 pre-mRNA SEQ ID NO. 9 Exon 21 pre-mRNA SEQ ID NO. 10 Exon 20xpre-mRNA Mouse SEQ ID NO. 11 SCN1A gene (NC_000068.7) SEQ ID NO. 12SCN1A pre-mRNA (encoding e.g., SCN1A mRNA NM_001313997.1) SEQ ID NO. 13Exon 21 gene SEQ ID NO. 14 Intron 21 gene SEQ ID NO. 15 Exon 22 gene SEQID NO. 16 Exon 21x gene SEQ ID NO. 17 Exon 21 pre-mRNA SEQ ID NO. 18Intron 21 pre-mRNA SEQ ID NO. 19 Exon 22 pre-mRNA SEQ ID NO. 20 Exon 21xpre-mRNA

TABLE 3 Sequences of target exon or intron in SCN1A pre-mRNA transcripts. SEQ IDSequence  NO. Type Sequence SEQ ID Exon 20 GUUUCAUUGGUCAGUUUAACAGCAAAUGCCUUGGGUUACUC NO. 7 pre-mRNAAGAACUUGGAGCCAUCAAAUCUCUCAGGACACUAAGAGCUCUGAGACCUCUAAGAGCCUUAUCUCGAUUUGAAGGGAUGAGG SEQ ID Intron 20 guaagaaaaaugaaagaaccugaaguauuguauauagccaaaauuaaacuaaauuaaauuuag NO. 8pre-mRNA aaaaaggaaaaucuaugcaugcaaaaggaauggcaaauucuugcaaaauugcuacuuuauuguuuuaucuguugcauauuuacuucuaggugauaugcaagagaaauaggccucucuugaaaugauauaauaucauuuaucugcugugcuuauuuaaaugacuuuauuuccuaauccaucuugggaguuuccuuacaaaucuauauacaaaaaaaagcugaugcauuauuaaaguacuauguguaaugauauaaugguaaucuaaaguaaauucuauaucagguacuuauucuuugugaugauauacuguacuuaacgaguuuuccugaaaauaaugugaaucacacaugugccuaaguaugaguguuaagaaaaaaaugaaaggaguuguuaaaacuuuugucuguauaaugccaaaguuugcauuauuugaauauauucaagauuagaugguuagauauuaaguguugacugaauuuauaaaacuaguaauacuaacuuaaagauuacauacaaauccacaucauuuuuauaacaauaaaguaaaacacuuauaaugaacagaaaauauaauuuugacucauuacuauagguaauuuauacauuaaccuuaacuugcaucuuauuggucagagucacacaaaauguuauuuuauccuuuucaaagaugcaauaaucauuuuccaucaugcauaacagauuagaaauuuugccauuauugacuuauuuuccaugccuuuuuuuacggcaugaagcauuaguuuauagauauauaauauaaaaaauuaguucugcuuuuuuuuaaaaaaaaauauuaucaaaacaaaacacugaauugugugauuccaauagaaaaacacugcucuuucaccuccuaagguguaguuacuuuuauggaaacuaagcuguauuguagacuuccauuugcacuuuguagauuguuuauagccuuauguucucuucucaagucuuauuauaaaugucacuuuguaagaacguaggacuugucuucgauuucccuaacauauaugaaaacuuuguccucauuaucgacaacucagaacaauauaauacaaguaguccucuuuuauuucucacagagagccucaaauuuucaccaaaauguuaacagaaauuaucucugggguguauaagaauuaagucuguuuuccaauuaaaugucacuuuguuuuguuucagacuggcaguuucaguucuggagaaaaaaaaugucauuuguguacauucuacuugaaaacauguugccugaaucaaaauaauauauuuuauauggcuugugaaaucugaacaaugcuaaacauuugaaaauauuauaaaccuuuuacauuugaccauuugaaaguuuauuaaauucauuggucaagugcucagauauuuccauacauuacacuucauuucuauaaaaaagcugaucuuaucgguauacuuuuaauuuucucagaaauaaccauaucuauaauuauuaaucaauaaugccuuuuauauuaaaagagguuaguuuuugaaacuuggaguuuuagacauaaaauccuuauaaaugcugauagugauauaacuaauaguuuaaauggucagauuuaugaauauggcucuauuccucauaaugacaacauacacacagcacuaaaaugacuaaucucuucaauacguguuuggcauuguagagucaaaauaacguuauaauugauucuauuuuuuauacuucuaguguuuggauauuuuauuuuguaaaaauauaaucaugaaugauggugagguuggauauaagaaugaugauuaugauugggaagugagauuugaacaugcucagaaacucucauuuaauucuuugcccuagcagcauaaaaucacaauagcugcgucaaagcguaacucaggcacucauuuuauuuuuguuguucuguuauuuuuucaaagcaugugcuuuuaugcaacauuacugaauaaagcauguuguacagugcuugauaagaaguuagaaaguaacaaauaaauuaucaucacguugcacuuuguguuuugcauguuuuaugcacauuucuggcugacagcuuuuaaacauuuauuguauuucaaauuuccaguccaaauuuuucaacuuguaaaauuaaacugagugaauugaugucgugaauaucuaggguaaaauaaaauuuguguuuaaauuuguauuuuuaauuuccuaaccuaggaaaucuuaaauaccuucuuuuucaaaagaacucaagucuuaauggauagggaaacagacggagagcaucaugaacaaaaaguaacaccaaauguucugucauaucagauuucuaacuaauaacaaacuauauauuucuauuuuguauaggauaaucuugcuccaacuuggaugggguggagcgcugguuccuccccugagcccuuuauuauggguacuguauuaccccuuuugcuaccuuuaauccuugcacugugacuuauguguaguggggugagggagggauugggaaggguacuauuauugcaccacaguagggaaaauacauuauuuacauccuaauccccucuuuucaauugucuuaaauuucauuugaaaaaaaaaaaaaccuuuaugaauuuacccucuguggauuuuaaccccaaugguugauaucuuuauuaaguuucauugaauaugauuuaguuauguguauauggaguuauccaucuuuggggagauuacuggauuggugagggcgggggacccugguguagaaugauuaugugaaaaaacaauuuaacuuguuaagcucaugauacuguuugaggcauacagccccugcuguuuaguacauuggucuggguccugaaaauuaccaguuagauaccaucaguugauuauugauauguaugagcagauacuagggugcaauauuucagguuucauaagacugguauugauugugaccacucucauuuuuuauuguguaaguucauaugggguuauuuucaaaauguuaacaaggcaaaaauauauuaagaaauaguugaauaagcacaugugaauuguguuguaaacaaaaaguuagaauaaaaaaauccacuuauuugaauuaugcagaauagaauacauaccuagaaauaaaacaaaaacgucuuaucaugaguauuaagauaaaauuuaaggcauaaacucacuucuuagaauaaguaacucccaacuaacuuucuaggauuuuaaaacauaacacagugaaaacauacauaaacauaacucuacauuuuauuuauucuuaaaguuuaaguguauuauacaagaagaagaguuuauauucgagagacagaaaaagucagaauuuuuguuuggaucaccaauauaucauagcuuacaaaaaaacugucuuaauuaaaacccacaacauaauuuuuuuagauuuuuaagaaagauucuauuauucuucuuuauacuuaaaaauggaugauuccuacuuugcccacuuuuauuuuuauucacauagauuuucuuuauuucuauuagagaagcacuagaauucaugaauaguguugauuugaaguucaaaguaauuaauucagauaaaaagacauuucugcauguaugaaaauuucuaaugugaauuugcauauuuaauuaucaauccuucauuuaguguagacuuauuuuuaaaaaugcagguaaugaaccagaaauagaauugguugugcuagaguagagaaacuuuauuugaugauuguuuugaaaaaaaagcuucugagaagaaacaaccucuaguacaguauuaauucauuaagauagcuccuuucucagacauuuccuuucauguagccugaaaguucaauuugaaauuuguucuuuccaauuuauucagacuaauucugccuacuuucuuccccccauaagaaccaauuacugcagcuuuauugagacugaaaaaaguuaauacaccuccuucuuugcugaaccaaggaauggcuuggaacucuugggaaaagacaaucuuuucuaugaucuuucauugucuaauuuaauacaucauaaauaugacuauagcuuuguauaauaaacuccccaauacugugccagauguuuucuaagauaaaguuauuuuauguucacaaaaaaaauaaaacuuuucucugggccaaauguaugccaacuuugcaaaucauauccugaagugcacugcugcagaguacaugcuugcgucauaaauuccauagaguucgcuuuaacucuaaaucaauccccaguuucaaaguaaaccucucaaacauauuaccuaagcacaaacuucucccugugcucaguuccuuaauuauucucaucccauauucagaaauaacauuuaaaaauuaugcuuugaucaauaaauacuaaucuaaacuuugcuucauuaacccauucauuuuugucaaccauuauuuuauuccuauauucaaagcucucugguauguucuuauauucaagacacucaaggcccuggaagauucacgaacauauguuugcaucuuaaauuuuuagaaaaucuuacaaucugucaggauuacacugaacucuaguacagaguaauauggguaccagauaagugggagcaacucuuccacguagacuggaaacagcacuaaaugcuauuuauaggcuacuuucugaacuuaacuuguuuuaaccucauuuuucucauaugccaaaugagaacgcaauacugaauuaucuguacaguucuguucaguacuagaauucugauucuugaauucaaaggggaaaacauuccucuuuauuuuggaggcuaaacugggggacaaaguuaggcuccaugaaagaagugcuauuugaacuaaagccuuuaagaggggagaguauuucagaagaggagcuauuagacaaggaauuucaauguaaauggcaucucaaucaccuggcaauuauauuagcacacgguuauuauauuaauugaaguggcaugaaguauagaugaccagggaaguuaaaacuggaaauauagauuguggagugaugugaauaccaagguaagaaaaauauuuguuaguuaccagagagccaauaaauaacuuucaagugggacuuggggaagauuaauucaucuuacauagauuaaaugaaggagaagguuaggagacagaugacagugcaaguaugaaauaacagagggcaguucuagguggugacugugagaauggaaaagagguggcaaagcugagaaacguuucaaagaaaaaaugugagacagguaaugugaaaagaaaaucgagaaauagguauagauaaucaguguucugcucauacucuaaauuggguguugaaggcaaaauacguauuuuaauuaguacucuguguauacacacuagaaacagcauuguaaucuggauaguggacaaaauauucagaaaagaagggaaauaguaacuugauuucaauuuccaaaucucuaaucugaaagaaaucuaauucuauucauccauuuaaaauaaauuauauaacgagaauuuaugaaguccauuguauuaaugcagacagucagaugagauaaggcaaagugucacgugucagcuugguaguugcaucggccacaucauuugguucugccuggauaacucaaccaaauuaauuuuucauacucauccccuccaccuuugucauuacugguauucuuauuuucuuuggcccacuuaucacacuguuuuauguuccccagaaggccuagaguucuuuacaggcuuuaaacagggaucagaaguauaagaaauuggcucauguauuuuuuuuucagacaggcaguuaaaaaaaauuguucuaaaaauacacuggcaucaaauggcaaauagaagauguuuugacgacuacuuccauuggaucagacugacaagaauaauacaagcacauagguggaauuaaacuuagcuauuaauguccaaguuugaggcagcugccccuuauaagcauuuuagggucuguuuuuagcuucccucuuagccacuccugugcagcuccagugggagguauggaggaaaaagcaaggaagccaucccuauguuguuuccaaacaugaacacucaagauuuuuaacuagugguccagaaguaaagagggggaaaacauccuucuauagaaaaaaaaaaaaguagauaauaauugaacacagaacuucaugugaucacaucagauuugagaacuauguauggcaucccucuuuuucuuauuuuccuaagaaaugauuucuauuauguuucauuugaaauaaguuuuuugaauuaaacucaguaaaugaaacaacugacaugacuggagcuugaaauaaacgaugugaugaucuaaugaaauacauaaugcaaauugucuugcuucuuaugcaaaaauuauuagucauagcaaugcaugaauaauuaaagacaauuauauuagguauuuaauaauauuuuuuauauuuaucaucugaauuuuuaaguuauuuuaaaaauauauuggucaaaucaacucagguccaaauguuuuaguuuuguucuuuaauauauugccuuuuuaaaaugaguuaaacuucuguauaggcuuuuuaacuuuucuuuauucugauaacacaauucugacuucaucuggcagcaaguuccucugauuuuccuuuuccuuuaaccuuuuaaugcuucucccucccuuuuuuuuaaaaacauuuuuguuucauuucuugguuauauugccuauaguuguuuuccuaaguguauugcuuaagaaaaaaaaaugaauuuuaagauuuuuuugaaccuugcuuuuacauauccuagaauaaauagcauugauagaaaaaaagaauggaaagaccagagauuacuaggggaauuuuuuuucuuuauuaacagauaagaauucugacuuuucuuuuuuuccauuuguguauuag SEQ ID Exon 21 gauaaucuugcuccaacuuggaugggguggagcgcugguuccuccccugagcccuuuauuau NO. 9pre-mRNA gg SEQ ID Exon 20x  GUGGUUGUGAAUGCCCUUUUAGGAGCAAUUCCAUCCAUCAUNO. 10 pre-mRNA GAAUGUGCUUCUGGUUUGUCUUAUAUUCUGGCUAAUUUUCAGCAUCAUGGGCGUAAAUUUGUUUGCUGGCAAAUUCUACCACUGUAUUAACACCACAACUGGUGACAGGUUUGACAUCGAAGACGUGAAUAAUCAUACUGAUUGCCUAAAACUAAUAGAAAGAAAUGAGACUGCUCGAUGGAAAAAUGUGAAAGUAAACUUUGAUAAUGUAGGAUUUGGGUAUCUCUCUUUGCUUCAAGUU SEQ ID Exon 21 GUUUCAUUGGUCAGUUUAACAGCAAAUGCCUUGGGUUACUC NO. 17 pre-mRNAUGAACUCGGGGCCAUCAAAUCCCUAAGGACACUAAGAGCUCUGAGACCCCUAAGAGCCUUAUCACGAUUUGAAGGGAUGAGG SEQ ID Intron 21guaagaaaaaggaaaacucugcagcguuguauauugucaaagcuaggcugaguucaacuuaac NO. 18pre-mRNA uaacgaaaaacacgugcaugcaaaaggaauggcaacccuuugcaaacuugcuacuuuacccuuuucucuguugcauauuuacuucuuggugauaugcaagagaaaaucggccucuuugaaaaugauuuaauaucauuuaucugcuuugcuaauuaaaaugaccuuaguucauaaucgaucuugggaguuuccuuauaauuccuaauacaaagggggaggggcagauacucucuuaaagaacuaaguugagucauguaauaauuaccuagagauaauuuuguuucauaucguucuccucuaugacagcc caucaguacuuaagggauccuauggaaaguaaugugaaucacaaauguguaugaauacaaaggaaaaaaugaagaauuguuaaauguuuugucuuuacaaugccaaaauucucauuauuugaauauauccaagggcagauauuaaccauugacuggaguauaauaauacugccucaacuguaacuaaauuaaugacauugaauaaguaagacacuaauuuaauuacuauaaauacauacacaucuuaugacaauacagcugauaaggaaaaagaacauguauuuuuauucauugccauacauggcucgucaaccuuaacuuaaaccucgguucucaguuacacagaguuuuauguugcucuuuugagcaaagcauuauuccccucuccauaauucaacacguaucagauuuuugcauuuauuggcucauuguuaugaugauuauuucaaagcauuauacaaucauuuauagaagaugugccgugugaaaauuauuuuuuuauuaagauccaaaauuuacgcucuuuaaaccaaucagaugaaauguauaaggcaaagagugcucauuguccugacacuuacaaaccaaggcuccaacaaacaggcuccucucugcaccacauagagggcuuucagccuguguccccccauaaaaaccuauuauaaauuuuauuauacuauacuguaagaaaccuguccaauuuuuaauuucucuagcacauaugaaaacuucucuucaguagauccaaguaagcacaaaggagcuuugauucucacacaagaaaucacauuuguauuaaaaauguaucauaaauuuucucccaauuaugcaaaacuuaaaugcuuuuccaauuaaaagagcacuuucguuucagaauagcaauuucaguugucaaggaaaacauuguuuuuauacauuuuauauaaaaauacaugagcuaaauuuaaauucacauuuuucaacuuuuuaugguuuuuuuauguuucuuuuucuuucgcauuuuuuaacaauccagccauuaccucuccucccugucccccuccuauaguuucucaucucauuccuccucccccuugccucugagaggaugcucccucccucacuaggccucccucuuccccagggcuucaaguuccucaaggauuauacacaucuucucccacugagaccaggccaggcaguccucugcuucugcccagccuguguauguuccugcuugguagcucagucucuggaagcucccuggggucuggguuaguugggacugcuggucuuccuauggaguuacccuccccuucaacuucuucaauccuuccccuaauucaaccacagguauccagacuucaguccaaugguugaguguaaauauuggcaucugucucugucagcuguuguuaggggcucagaggacagccaugcuaggcuccugccuacaagcagcacaccauaacaucaguaauagugucaggccuuuaaugaauaaugcuacguauauaagguuguuagauuauacuucaacuuugaucuuuuagaauauuauuaaauccagucguuuauuuuuuauauguaauauugacuuuccauaacaaaugagucuauuuuccuuuuguguagaaauaacuuuaucaauuauuguuaauaaugcuuuugucaauuauuguugauaugcuucucuuuuuuaaaacuggagucaucaaaacaaaaauucuggguagauauuacgaagcugacuccuuggucagcuuggcacauagugagaccacaaauaucucaaggucacggcaauuccucaccaccaguuuggcauugugaagucaaaaccaaccuucuguugauucugguuuuuguauuucuaguaugagacauuuucuacuuuguaagaguauauaacuguggauggcggcgagguugggcaugaugaugauuguaagcgggaagugagcuagaguaucuucagaaacucucacuuuauccuccuuggcagcauagaaccgcaaucgcuguguccgagugucaaccaggcagucauuuuguuuuggguuuuuugucacucuuucaaagcaugugcuucuacgcaacacuaccaaacacagcaugcugcauagugcuugagaaggaguuagaaaguaacaaacgaguuaucaucacguugcccuuuguguuuugcaugucuuaugcacacuuuuggcugacagcuuuugaacauuuauuguauuucaaauuuccaguccaaauuuuuuuuucaacuugugaaauugaacggaaugaaccgaugucgugaauaccuagggucaaauaaaacuuguauuuaaauucguaguuuuaauuucccaagcugggaaaaucguaaaaaccuuuuccaaagaacucaagucuuaguugcuagggaaacaggcagugagcaucauauacaaaaaguaacaccaaauguucugucauaucagcuuucuaacuaauaauaaacuauauauuucuauuuuauauaggauaaucuugcuccaacuuggaugggguggagcggugguuccuccccucagcccuuuauuauggguacuguauuaccccuuuugcuaccuuuaauccuugcacugugacuuauguguagugggauugagggagggagugggaaggguacaauugcaccacaguagggacaauacaggauuuauuuccaaauccacuacuuuuaaugagcuuaaacuucuuuugggaaaaaaaaaguuaucucugacuuaccaucuguggauuauaaccccagaaguacauaucuuuauuacguuucacugaauaugauuuagcuauuuauacuucauuguccauuuauggggaaauuacucaauuggugagggugggggacccugguguaggaugcugaugaaaacguuuucauuugucaagcucaugguagugacagagcauauaguccuuauuuuuucaacacacugcucuggucccucaaugggccagucacauuccaucaguugaucguugaugugugcgagcaguggcuaaagguacaacaggccagguaucucaggguugccaaugguuaugaucauucucaucuuuauugcauaaaaauguguuauuugcagaaaguagcaaggcaagaucccugugaaacaagggaauacaaaaaaaaaaaaagaugugcuuuaaguuauaaaaccaaaacaugugaaaagucaacuucauugaaguauaaagaauaggauaugcaugaaaaacaaaaaaaucaugagcacuaagaaauugguguauaagccaacuccuuguaagcuaccccaauuaacuucccagaaucuuagaaggcaucacagugcaccccaaaauaaaaagccaaacugacacuucugcuuccucuuaaaauguaggagucuuggauaagaaagauaauuuuuauuguuuggaagaaaaaaaaauuguuuggauaauugaggcauuuaucuaucaaaaauauuuaucuuaauaaaauuucacaacacugauuuaguuguuggcuuuucuaaaaauuuuuuauauuucauauuaagaacucaugauuuuuacuuuccauuuuuuaaauucuuauucacauaugguuuuucucuauuucuuagaaaagcuauagaacccaugguuuccggugacuuaaaaaacuaaucuaaaugucuucacuuagaugauacuuucaaaugcacugaaauuucuaauaucaacaagaauauuugccugguccuaauuuuucacugauuuaauaaaaaguaugaacccuaaagaagaaauagacuugaagaacugguugugugacaauacagaaauucugcugggagauguccuuuuaaaacauuguuagaagagacaaccucuacaauccacccauuaagcauacuucucucuuagacaucuccuuuuauguaccuuauaaccucaauguguucuuuccaauugacuuagaccaacacuucccagcgcaucccacaugggagccaauuacugacucucccuagagacugcaaagaauuaauauuguagaaccaagggaugguugggcucuugggagaggcaauccguuugugaucuuuugcucuggauguuaaugaaaucgcaacuuuaaguggauuuucaguggcaaauccucugauccuaugccaaauguucucuaagacaaacauccuuguaaaauaaaugucucacugggccaaaucuauggcaaauuugcacguuuuccugaacugcauuccuauauaguauuugccauccugaauucacuaaugggcauuacuuuuaauucaaaaccagucccuucuucaaaggaaaucucucccauuuauuacauuaugcaaacugcuuucuuaugcagugguuaaauccuuagccaggcaaguaugaggacuggaauuuggauauccagaacccucagaaaugucggaugggcauaguagcuuacauguaauuccagagcuagaaagaugaaacuagcccuguccucaagcucugaauucaguuggugcaauaaauaagaaagaauccccccccccgaccccuacaucucuuuccucuacaucuauacauguauuucccauacagcucuaugcuuccacauauaugcucacauaugugcaugcacacuugcacacauauguacacuugacacauugaagcaucauaaucuaacaauuggaauauaagaaaauauuuaacuuucacacagagcagucagagaaaaccauaagagguugaaacucuugaaaaaacuugcaggauaaacagaaaagaguaugagaugccaauucuggucuacuuucugaaccuaacuuguuuuaccuucauuagucuaauuuuccaaaugaaccccaagcaccaaauuguccuuauugcucuuuccaguacuaaauuauaauuccucaauucgaaggcaaacacucucaucuuuguuaaggauguguagaguuagacucaauaaacgacauguauauuugagcuaaagccuggaggagggagauuauuucaagggcaaguacuuggcagggaguuucaaagaaagaaggcucucaauucucagacuaacaccuuagcguggagugacugucacagaggagggugaaguguaugucaccaguuagggaagcugaaaggggaaauguaucaggcuugugagaauccuucagcagccaagucuagcaccugagcacaauccccagaacugaccccacaugguggaaaggagaggaauaauuccugcaaauuuuccugugaccuccacccaagugcuauagaaaaugcaugcaugcccacaugcacacacaaauaaacauaguugcaaacuguuuugaggaaaauaaccucacaaacugucgagugauguaaaugccaaagaaagagaaauguuuucuaauggcuagagaaccauuaaggaauuuuucaaaaugggacaugggauagauaaauuuaguauccauacugaaagaaggcaagcaaauaaaaucugauaagaauguaauucuuaguaaccgaggacagagcgaagauagagaacagggccaauggccaagguggaaagguuugaaggaagcagcaugaaaccuacagacuuguaccaaaauguucagugucaugaguguuaaaagugaaaagcuugcauguuaguauggauuucauauccucggcagacagagcaccucacugugagugggagaugaaguauucagaaaugagaaacaacuacucaauuucaguguucauaucucaaauccaauaaacaacuuuagggguacaauuuuuuaaaaaauuacauuaaaauguuuuuaaaucucuucuuaauaauuuaaaaauuaaauugaaaauaacuuuaaaaaguaauauauacaggaaagccugugugcuaauuuuuuagggaggccauaaagggagauaguugcucauuaauuucuacacaucagccuaucuuuggcuucugccuugauagcgcacucugaauuaucuucuucauguucaucccucaucuuuauuguuacugguuucauuucccuuggccacauagcccacuauuuuguauuccccaauggauauuguuccuuacaaaguucagccagggcucagaaguacaaggaauuggcucuuauacuucugucagacaggcaaaaacuucuaaaauuauacuauaauaaaaaucaaagagaugauauucauaauuaaacuaacaaaaguggcaggcccccccucccccaacaugaguagaauuaaucugacguccauguucaagucugaaacacacuugccaauuaagagcacauuagggccagccuuuaucucccucuuaguuacuaaugugcaguucaauggugagcuauagagaaggaagccaagacuaccauaugucaaauauaaaaaaaaaaaaucccauuuuaaaucuguagucccgaauuaaggacaagagagagggaaauaucuuugacauuagaaaauggagaaaauauuuuagcacaggacuuuacucagucacaucagaguugauaaguacguaugacaucccucuuuuuccuguuuuccugagaaaaugaucucucuaguguuucauuuaagauaaguuuauugaauuaaacucaguaaaugaaacaacugacaugacuggagcuugaaauaaacgaugugaugaucuacugaaauacaugaugcuaaauugucuugcuucuuaugcaaaaacuacuauuaguuauagcaaugcauggauaauuaaggccaaaaauauauuagauguuaaaaauaguuuuauauuuauacaucugaauuuuaauuuauauuuaaaguauauugguccaaucaauucaugcccaaauguuuuaguucuauucuuugagauacuguuuuguuuugggauuuuuuuuuaugagcuaaucucuugccuaggaguuccuacuucucucuccuccuuuuauuuuuucuaauaaacuacacaugugucuucauccaggagcuaacuucucccauuuugcuuuuccuuuagcaccuuuuuuauauuagauuucuuucuuuucuccaucucuuugcauauugccuauauuucuuuuccuaagcauaauauuuaaaaaagacugaguuuuauguuaagauuauuucugcuuugcucuuacacagauaggauaaguagucuugauagaaaauaaaucaaugauuccuagggggaugucuuuuugcuuuuaaucaauaaggauucugacuucucuuucucuccauuuguguauuag SEQ ID Exon 22 gauaaucuugcuccaacuuggaugggguggagcggugguuccuccccucagcccuuuauuau NO. 19pre-mRNA gg SEQ ID Exon 21x  GUGGUUGUGAAUGCCCUGUUAGGAGCAAUUCCAUCCAUCAUNO. 20 pre-mRNA GAAUGUGCUUCUGGUUUGCCUUAUAUUCUGGCUAAUUUUCAGCAUCAUGGGCGUAAAUUUGUUUGCUGGCAAAUUCUACCACUGUGUUAACACCACAACUGGUGACAUAUUUGAGAUCAGCGAAGUCAAUAAUCAUUCUGAUUGCCUAAAACUAAUAGAAAGAAAUGAGACCGCCCGGUGGAAAAAUGUGAAAGUAAACUUUGAUAAUGUAGGAUUUGGGUAUCUUUCUUUGCUUCAAGUU

Example 2: Confirmation of NIE Via Cycloheximide Treatment

RT-PCR analysis using cytoplasmic RNA from DMSO-treated (CHX−) orcycloheximide-treated (CHX+) mouse Neuro 2A cells (FIG. 3A) and RenCellVM (human neuroprogenitor cells) (FIG. 3B) and primers in exon 20 andexon 23 confirmed the presence of a band corresponding to theNMD-inducing exon (20x). The identity of the product was confirmed bysequencing. Densitometry analysis of the bands was performed tocalculate percent exon 20x inclusion of total SCN1A transcript.Treatment of RenCell VM with cycloheximide (CHX+) to inhibit NMD led toa 2-fold increase of the product corresponding to the NMD-inducing exon20x in the cytoplasmic fraction (cf. light grey bar, CHX−, to dark greybar, CHX+).

Example 3: SCN1A Exon 20x Region ASO Walk

A graphic representation of the ASO walk performed for SCN1A exon 20xregion targeting sequences immediately upstream of the 3′ splice site,across the 3′splice site, exon 20x, across the 5′ splice site, anddownstream of the 5′ splice site using 2′-MOE ASOs, PS backbone, isshown in FIG. 4. ASOs were designed to cover these regions by shifting 5nucleotides at a time. A list of ASOs targeting SCN1A is summarized inTable 4. Sequences of ASOs are summarized in Table 5a and Table 5b andTable 6a and Table 6b.

TABLE 4 List of ASOs targeting SCN1A Gene Pre-mRNA ASOs SEQ ID NO. SEQID NO. SEQ ID NO. NIE SEQ ID NO. 1 SEQ ID NO. 2 SEQ ID NOs: Exon 20x21-67, 210-256 SEQ ID NO. 11 SEQ ID NO. 12 SEQ ID NOs: Exon 21x 68-114,257-303

TABLE 5  Sequences of ASOs targeting human SCN1A SEQ ID NO.Sequence name ASO sequence 21 SCN1A-IVS19X−81 GATGCTCTCCGTCTGTTT 22SCN1A-IVS19X−76 TTCATGATGCTCTCCGTC 23 SCN1A-IVS19X−71 TTTTGTTCATGATGCTCT24 SCN1A-IVS19X−66 TTACTTTTTGTTCATGAT 25 SCN1A-IVS19X−61TGGTGTTACTTTTTGTTC 26 SCN1A-IVS19X−56 ACATTTGGTGTTACTTTT 27SCN1A-IVS19X−51 ACAGAACATTTGGTGTTA 28 SCN1A-IVS19X−46 ATATGACAGAACATTTGG29 SCN1A-IVS19X−41 ATCTGATATGACAGAACA 30 SCN1A-IVS19X−36TAGAAATCTGATATGACA 31 SCN1A-IVS19X−31 TTAGTTAGAAATCTGATA 32SCN1A-IVS19X−26 TGTTATTAGTTAGAAATC 33 SCN1A-IVS19X−21 TAGTTTGTTATTAGTTAG34 SCN1A-IVS19X−16 ATATATAGTTTGTTATTA 35 SCN1A-IVS19X−11TAGAAATATATAGTTTGT 36 SCN1A-IVS19X−6 CAAAATAGAAATATATAG 37SCN1A-IVS19X−3 ATACAAAATAGAAATATA 38 SCN1A-IVS19X−1 CTATACAAAATAGAAATA39 SCN1A-I19X/E20X+2 TCCTATACAAAATAGAAA 40 SCN1A-I19X/E20X+4TATCCTATACAAAATAGA 41 SCN1A-I19X/E20X+6 ATTATCCTATACAAAATA 42SCN1A-Ex20X+1 AGTTGGAGCAAGATTATC 43 SCN1A-Ex20X+6 ATCCAAGTTGGAGCAAGA 44SCN1A-Ex20X+11 ACCCCATCCAAGTTGGAG 45 SCN1A-Ex20X+16 GCTCCACCCCATCCAAGT46 SCN1A-Ex20X+21 CCAGCGCTCCACCCCATC 47 SCN1A-Ex20X−24GAACCAGCGCTCCACCCC 48 SCN1A-Ex20X−19 GGGAGGAACCAGCGCTCC 49 SCN1A-Ex20X−3ATAATAAAGGGCTCAGGG 50 SCN1A-Ex20X−1 CCATAATAAAGGGCTCAG 51SCN1A-E20X/I20X−6 GTAATACAGTACCCATAA 52 SCN1A-E20X/I20X−4GGGTAATACAGTACCCAT 53 SCN1A-IVS20X+13 TTAAAGGTAGCAAAAGGG 54SCN1A-IVS20X+18 AAGGATTAAAGGTAGCAA 55 SCN1A-IVS20X+23 AGTGCAAGGATTAAAGGT56 SCN1A-IVS20X+28 GTCACAGTGCAAGGATTA 57 SCN1A-IVS20X+33CATAAGTCACAGTGCAAG 58 SCN1A-IVS20X+38 CTACACATAAGTCACAGT 59SCN1A-IVS20X+43 CCCCACTACACATAAGTC 60 SCN1A-IVS20X+48 CCTCACCCCACTACACAT61 SCN1A-IVS20X+53 CCCTCCCTCACCCCACTA 62 SCN1A-IVS20X+58CCAATCCCTCCCTCACCC 63 SCN1A-IVS20X+63 CCTTCCCAATCCCTCCCT 64SCN1A-IVS20X+68 AGTACCCTTCCCAATCCC 65 SCN1A-IVS20X+73 ATAATAGTACCCTTCCCA66 SCN1A-IVS20X+78 GTGCAATAATAGTACCCT 67 SCN1A-IVS20X+83CTGTGGTGCAATAATAGT

TABLE 5b  Sequences of ASOs targeting human SCN1A SEQ ID NO.Sequence name ASO sequence 210 SCN1A-IVS19X−81 GAUGCUCUCCGUCUGUUU 211SCN1A-IVS19X−76 UUCAUGAUGCUCUCCGUC 212 SCN1A-IVS19X−71UUUUGUUCAUGAUGCUCU 213 SCN1A-IVS19X−66 UUACUUUUUGUUCAUGAU 214SCN1A-IVS19X−61 UGGUGUUACUUUUUGUUC 215 SCN1A-IVS19X−56ACAUUUGGUGUUACUUUU 216 SCN1A-IVS19X−51 ACAGAACAUUUGGUGUUA 217SCN1A-IVS19X−46 AUAUGACAGAACAUUUGG 218 SCN1A-IVS19X−41AUCUGAUAUGACAGAACA 219 SCN1A-IVS19X−36 UAGAAAUCUGAUAUGACA 220SCN1A-IVS19X−31 UUAGUUAGAAAUCUGAUA 221 SCN1A-IVS19X−26UGUUAUUAGUUAGAAAUC 222 SCN1A-IVS19X−21 UAGUUUGUUAUUAGUUAG 223SCN1A-IVS19X−16 AUAUAUAGUUUGUUAUUA 224 SCN1A-IVS19X−11UAGAAAUAUAUAGUUUGU 225 SCN1A-IVS19X−6 CAAAAUAGAAAUAUAUAG 226SCN1A-IVS19X−3 AUACAAAAUAGAAAUAUA 227 SCN1A-IVS19X−1 CUAUACAAAAUAGAAAUA228 SCN1A-I19X/E20X+2 UCCUAUACAAAAUAGAAA 229 SCN1A-I19X/E20X+4UAUCCUAUACAAAAUAGA 230 SCN1A-I19X/E20X+6 AUUAUCCUAUACAAAAUA 231SCN1A-Ex20X+1 AGUUGGAGCAAGAUUAUC 232 SCN1A-Ex20X+6 AUCCAAGUUGGAGCAAGA233 SCN1A-Ex20X+11 ACCCCAUCCAAGUUGGAG 234 SCN1A-Ex20X+16GCUCCACCCCAUCCAAGU 235 SCN1A-Ex20X+21 CCAGCGCUCCACCCCAUC 236SCN1A-Ex20X−24 GAACCAGCGCUCCACCCC 237 SCN1A-Ex20X−19 GGGAGGAACCAGCGCUCC238 SCN1A-Ex20X−3 AUAAUAAAGGGCUCAGGG 239 SCN1A-Ex20X−1CCAUAAUAAAGGGCUCAG 240 SCN1A-E20X/I20X−6 GUAAUACAGUACCCAUAA 241SCN1A-E20X/I20X−4 GGGUAAUACAGUACCCAU 242 SCN1A-IVS20X+13UUAAAGGUAGCAAAAGGG 243 SCN1A-IVS20X+18 AAGGAUUAAAGGUAGCAA 244SCN1A-IVS20X+23 AGUGCAAGGAUUAAAGGU 245 SCN1A-IVS20X+28GUCACAGUGCAAGGAUUA 246 SCN1A-IVS20X+33 CAUAAGUCACAGUGCAAG 247SCN1A-IVS20X+38 CUACACAUAAGUCACAGU 248 SCN1A-IVS20X+43CCCCACUACACAUAAGUC 249 SCN1A-IVS20X+48 CCUCACCCCACUACACAU 250SCN1A-IVS20X+53 CCCUCCCUCACCCCACUA 251 SCN1A-IVS20X+58CCAAUCCCUCCCUCACCC 252 SCN1A-IVS20X+63 CCUUCCCAAUCCCUCCCU 253SCN1A-IVS20X+68 AGUACCCUUCCCAAUCCC 254 SCN1A-IVS20X+73AUAAUAGUACCCUUCCCA 255 SCN1A-IVS20X+78 GUGCAAUAAUAGUACCCU 256SCN1A-IVS20X+83 CUGUGGUGCAAUAAUAGU

TABLE 6a  Sequences of ASOs targeting mouse SCN1A SEQ ID NO.Sequence name ASO sequence 68 mScn1a-IVS20X−81 GATGCTCACTGCCTGTTT 69mScn1a-IVS20X−76 TATATGATGCTCACTGCC 70 mScn1a-IVS20X−71TTTTGTATATGATGCTCA 71 mScn1a-IVS20X−66 TTACTTTTTGTATATGAT 72mScn1a-IVS20X−61 TGGTGTTACTTTTTGTAT 73 mScn1a-IVS20X−56ACATTTGGTGTTACTTTT 74 mScn1a-IVS20X−51 ACAGAACATTTGGTGTTA 75mScn1a-IVS20X−46 ATATGACAGAACATTTGG 76 mScn1a-IVS20X−41AGCTGATATGACAGAACA 77 mScn1a-IVS20X−36 TAGAAAGCTGATATGACA 78mScn1a-IVS20X−31 TTAGTTAGAAAGCTGATA 79 mScn1a-IVS20X−26TATTATTAGTTAGAAAGC 80 mScn1a-IVS20X−21 TAGTTTATTATTAGTTAG 81mScn1a-IVS20X−16 ATATATAGTTTATTATTA 82 mScn1a-IVS20X−11TAGAAATATATAGTTTAT 83 mScn1a-IVS20X−6 TAAAATAGAAATATATAG 84mScn1a-IVS20X−3 ATATAAAATAGAAATATA 85 mScn1a-IVS20X−1 CTATATAAAATAGAAATA86 mScn1a-I20X/E21X+2 TCCTATATAAAATAGAAA 87 mScn1a-I20X/E21X+4TATCCTATATAAAATAGA 88 mScn1a-I20X/E21X+6 ATTATCCTATATAAAATA 89mScn1a-Ex21X+1 AGTTGGAGCAAGATTATC 90 mScn1a-Ex21X+6 ATCCAAGTTGGAGCAAGA91 mScn1a-Ex21X+11 ACCCCATCCAAGTTGGAG 92 mScn1a-Ex21X+16GCTCCACCCCATCCAAGT 93 mScn1a-Ex21X+21 CCACCGCTCCACCCCATC 94mScn1a-Ex21X-24 GAACCACCGCTCCACCCC 95 mScn1a-Ex21X-19 GGGAGGAACCACCGCTCC96 mScn1a-Ex21X-3 ATAATAAAGGGCTGAGGG 97 mScn1a-Ex21X-1CCATAATAAAGGGCTGAG 98 mScn1a-E21X/I21X-6 GTAATACAGTACCCATAA 99mScn1a-E21X/I21X-4 GGGTAATACAGTACCCAT 100 mScn1a-IVS21X+13TTAAAGGTAGCAAAAGGG 101 mScn1a-IVS21X+18 AAGGATTAAAGGTAGCAA 102mScn1a-IVS21X+23 AGTGCAAGGATTAAAGGT 103 mScn1a-IVS21X+28GTCACAGTGCAAGGATTA 104 mScn1a-IVS21X+33 CATAAGTCACAGTGCAAG 105mScn1a-IVS21X+38 CTACACATAAGTCACAGT 106 mScn1a-IVS21X+43TCCCACTACACATAAGTC 107 mScn1a-IVS21X+48 CTCAATCCCACTACACAT 108mScn1a-IVS21X+53 CCTCCCTCAATCCCACTA 109 mScn1a-IVS21X+58CACTCCCTCCCTCAATCC 110 mScn1a-IVS21X+63 CTTCCCACTCCCTCCCTC 111mScn1a-IVS21X+68 GTACCCTTCCCACTCCCT 112 mScn1a-IVS21X+73CAATTGTACCCTTCCCAC 113 mScn1a-IVS21X+78 TGGTGCAATTGTACCCTT 114mScn1a-IVS21X+83 TACTGTGGTGCAATTGTA

TABLE 6b  Sequences of ASOs targeting mouse SCN1A SEQ ID NO.Sequence name ASO sequence 257 mScn1a-IVS20X−81 GAUGCUCACUGCCUGUUU 258mScn1a-IVS20X−76 UAUAUGAUGCUCACUGCC 259 mScn1a-IVS20X−71UUUUGUAUAUGAUGCUCA 260 mScn1a-IVS20X−66 UUACUUUUUGUAUAUGAU 261mScn1a-IVS20X−61 UGGUGUUACUUUUUGUAU 262 mScn1a-IVS20X−56ACAUUUGGUGUUACUUUU 263 mScn1a-IVS20X−51 ACAGAACAUUUGGUGUUA 264mScn1a-IVS20X−46 AUAUGACAGAACAUUUGG 265 mScn1a-IVS20X−41AGCUGAUAUGACAGAACA 266 mScn1a-IVS20X−36 UAGAAAGCUGAUAUGACA 267mScn1a-IVS20X−31 UUAGUUAGAAAGCUGAUA 268 mScn1a-IVS20X−26UAUUAUUAGUUAGAAAGC 269 mScn1a-IVS20X−21 UAGUUUAUUAUUAGUUAG 270mScn1a-IVS20X−16 AUAUAUAGUUUAUUAUUA 271 mScn1a-IVS20X−11UAGAAAUAUAUAGUUUAU 272 mScn1a-IVS20X−6 UAAAAUAGAAAUAUAUAG 273mScn1a-IVS20X−3 AUAUAAAAUAGAAAUAUA 274 mScn1a-IVS20X−1CUAUAUAAAAUAGAAAUA 275 mScn1a-I20X/E21X+2 UCCUAUAUAAAAUAGAAA 276mScn1a-I20X/E21X+4 UAUCCUAUAUAAAAUAGA 277 mScn1a-I20X/E21X+6AUUAUCCUAUAUAAAAUA 278 mScn1a-Ex21X+1 AGUUGGAGCAAGAUUAUC 279mScn1a-Ex21X+6 AUCCAAGUUGGAGCAAGA 280 mScn1aEx21X+11 ACCCCAUCCAAGUUGGAG281 mScn1a-Ex21X+16 GCUCCACCCCAUCCAAGU 282 mScn1a-Ex21X+21CCACCGCUCCACCCCAUC 283 mScn1a-Ex21X-24 GAACCACCGCUCCACCCC 284mScn1a-Ex21X-19 GGGAGGAACCACCGCUCC 285 mScn1a-Ex21X-3 AUAAUAAAGGGCUGAGGG286 mScn1a-Ex21X-1 CCAUAAUAAAGGGCUGAG 287 mScn1a-E21X/I21X-6GUAAUACAGUACCCAUAA 288 mScn1a-E1X/I21X-4 GGGUAAUACAGUACCCAU 289mScn1a-IVS21X+13 UUAAAGGUAGCAAAAGGG 290 mScn1a-IVS21X+18AAGGAUUAAAGGUAGCAA 291 mScn1a-IVS21X+23 AGUGCAAGGAUUAAAGGU 292mScn1a-I21X+28 GUCACAGUGCAAGGAUUA 293 mScn1a-IVS21X+33CAUAAGUCACAGUGCAAG 294 mScn1a-IVS21X+38 CUACACAUAAGUCACAGU 295mScn1a-IVS21X+43 UCCCACUACACAUAAGUC 296 mScn1a-IVS21X+48CUCAAUCCCACUACACAU 297 mScn1a-IVS21X+53 CCUCCCUCAAUCCCACUA 298mScn1a-IVS21X+58 CACUCCCUCCCUCAAUCC 299 mScn1a-IVS21X+63CUUCCCACUCCCUCCCUC 300 mScn1a-IVS21X+68 GUACCCUUCCCACUCCCU 301mScn1a-IVS21X+73 CAAUUGUACCCUUCCCAC 302 mScn1a-IVS21X+78UGGUGCAAUUGUACCCUU 303 mScn1a-IVS21X+83 UACUGUGGUGCAAUUGUA

Example 4: SCN1A Exon 20x Region ASO Walk Evaluated by RT-PCR

ASO walk sequences can be evaluated by for example RT-PCR. In FIG. 5A, arepresentative PAGE shows SYBR-safe-stained RT-PCR products of SCN1Amock-treated (Sham), SMN-control ASO treated (SMN), or treated with a2′-MOE ASO targeting the exon 20x region as described herein in theExample 3 and in the description of FIG. 4, at 20 μM concentration inRenCell VM cells by gymnotic uptake. Two products corresponding to exon20x inclusion (top band) and full-length (exon 20x exclusion, bottomband) were quantified and percent exon 20x inclusion is plotted in thebar graph (FIG. 5B). The black line indicates no change with respect toSham. The full-length products were also normalized to RPL32 internalcontrol and fold-change relative to Sham is plotted in the bar graph(FIG. 5C). The black line indicates a ratio of 1 and no change withrespect to Sham.

Example 5: SCN1A Exon 20x Region ASO Walk Evaluated by RT-qPCR

SYBR-green RT-qPCR SCN1A amplification results normalized to RPL32,obtained using the same ASO uptake experiment that were evaluated bySYBR-safe RT-PCR as shown in FIG. 6, are plotted as fold change relativeto Sham confirming the SYBR-safe RT-PCR results. The black lineindicates a ratio of 1 (no change with respect to sham).

Example 6: Dose-Dependent Effect of Selected ASO in CXH-Treated Cells

In FIG. 8A, a representative PAGE shows SYBR-safe-stained RT-PCRproducts of mouse Scn1a mock-treated (Sham, RNAiMAX alone), or treatedwith Ex21x+1 2′-MOE ASO targeting the exon 21x (mouse nomenclature,corresponds to human exon 20x), at 30 nM, 80 nM, and 200 nMconcentrations in Neuro 2A (mouse neuroblastoma) cells by RNAiMAXtransfection. Ex21x+1 (mouse nomenclature) and Ex20x+1 (humannomenclature) are identical. Two products corresponding to exon 21xinclusion (top band) and full-length (exon 21x exclusion, bottom band)were quantified and percent exon 21x inclusion is plotted in the bargraph (FIG. 8B). The full-length products were also normalized to HPRTinternal control and fold-change relative to Sham is plotted in the bargraph (FIG. 8C). The black line indicates a ratio of 1 and no changewith respect to Sham.

Example 7: Intravitreal (IVT) Injection of Selected ASOs

FIG. 9A shows PAGEs of SYBR-safe-stained RT-PCR products of mouse Scn1afrom PBS-injected (1 μL) left eye (−) or IVS20x-21, Ex21x+1, IVS21x+18,IVS21x+33 or Cep290 (negative control ASO; Gerard et al, Mol. Ther. Nuc.Ac., 2015) 2′-MOE ASO-injected (1 μL) right eye (+) at 10 mMconcentration. Ex21x+1, IVS21x+18, and IVS21x+33 (mouse nomenclature)and Ex20x+1, IVS20x+18, and IVS20x+33 (human nomenclature) areidentical. Two products corresponding to exon 21x inclusion (top band)and full-length (exon 21x exclusion, bottom band) were quantified andpercent exon 21x inclusion is plotted in FIG. 9B. White bars correspondto ASO-injected eyes and grey bars correspond to PBS-injected eyes, n=5in each group. The full-length products were normalized to GAPDHinternal control and fold-change of ASO-injected eye relative toPBS-injected eye is plotted in FIG. 9C. The black line indicates a ratioof 1 and no change with respect to PBS, n=5 in each group.

Example 8: Intracerebroventricular (ICV) Injection of Selected ASOs

FIG. 10A shows PAGEs of SYBR-safe-stained RT-PCR products of mouse Scn1afrom uninjected (-, no ASO control), or 300 μg of Cep290 (negativecontrol ASO; Gerard et al, Mol. Ther. Nuc. Ac., 2015), Ex21x+1,IVS21x+18, IVS21x+33 2′-MOE ASO-injected brains. Ex21x+1, IVS21x+18, andIVS21x+33 (mouse nomenclature) and Ex20x+1, IVS20x+18, and IVS20x+33(human nomenclature) are identical. Two products corresponding to exon21x inclusion (top band) and full-length (exon 21x exclusion, bottomband) were quantified and percent exon 21x inclusion was plotted in thebar graph in FIG. 10B, n=6 (each targeting ASO), n=5 (Cep290 ASO), n=1(uninjected, no ASO control). Taqman PCR was performed using twodifferent probes spanning exons 21 and 22 junction and the products werenormalized to GAPDH internal control and fold-change of ASO-injectedrelative to Cep290-injected brains was plotted in the bar graph in FIG.10C. The black line indicates a ratio of 1 and no change with respect toCep290, n=6 (each targeting ASO), n=5 (Cep290 ASO), n=1 (uninjected, noASO control).

FIGS. 11A-C depict exemplary dose-dependent response from ICV injectionof selected ASOs in C57BL6J mice (male, 3 months old). FIG. 11A showsPAGE gels of SYBR-safe-stained RT-PCR products of mouse Scn1a from 300ug of Cep290 (negative control ASO; Gerard et al, Mol. Ther. Nuc. Ac.,2015), or 33 ug, 100 ug, and 300 ug of Ex21x+1 2′-MOE ASO-injectedbrains. Ex21x+1 (mouse nomenclature) and Ex20x+1, (human nomenclature)are identical. Two products corresponding to exon 21x inclusion (topband) and full-length (exon 21x exclusion, bottom band) were quantified.FIG. 11B depicts a graph plotting the percent exon 21x inclusion fromthe data in FIG. 11A, n=5 (each group). FIG. 11C depicts a graph fromresults of a Taqman qPCR assay performed using two different probesspanning exons 21 and 22 junction. The products were normalized to Gapdhinternal control and fold-change of ASO-injected relative toCep290-injected brains is plotted. The black line indicates a ratio of 1and no change with respect to Cep290, n=5 (each group).

FIGS. 12A-C depict exemplary results from ICV injection of a selectedASO in C57BL6J mice (postnatal day 2). FIG. 12A shows PAGE gels ofSYBR-safe-stained RT-PCR products of mouse Scn1a from uninjected (-, noASO control), or 20 μg Ex21x+1 2′-MOE ASO-injected brains are shown. Twoproducts corresponding to exon 21x inclusion (top band) and full-length(exon 21x exclusion, bottom band) were quantified. Ex21x+1 (mousenomenclature) and Ex20x+1 (human nomenclature) are identical. FIG. 12Bdepicts a graph plotting the percent exon 21x inclusion from the data inFIG. 12A, n=4 (each group). FIG. 12C depicts a graph from results of aTaqman qPCR assay performed using two different probes spanning exons 21and 22 junction. The products were normalized to Gapdh internal controland fold-change of ASO-injected relative to no-ASO-control brains isplotted. The black line indicates a ratio of 1 and no change withrespect to no-ASO control, n=4 (each group).

Example 9: Targeted Augmentation of Nuclear Gene Output for theTreatment of Dravet Syndrome

Dravet syndrome (DS) is a devastating childhood genetic diseasecharacterized by severe seizures, cognitive & motor impairments anddeath. The primary cause of DS is decreased expression of the sodiumvoltage-gated channel type 1 alpha subunit (Nav1.1). SCN1Anon-productive splicing event is conserved between human and mouse. FIG.13A depicts a graph plotting the percent exon 21x inclusion in theindicated mouse CNS samples. FIG. 13B depicts a graph plotting thepercent exon 20x inclusion in the indicated human CNS samples. In thisstudy, an antisense oligonucleotides (ASO) therapy was utilized toincrease productive Scn1a mRNA and consequently restore levels ofNa_(v)1.1 protein.

FIG. 14A depicts a graph plotting the percent decrease in exon 21xinclusion at the indicated doses (n=3-6 per group). FIG. 14B depicts agraph plotting the percent increase in Scn1a mRNA at the indicated doses(n=3-6 per group). FIG. 14C depicts a graph plotting the percentincrease in Nav 1.1 protein levels at the indicated doses (n=2 pergroup).

FIG. 15A depicts a graph plotting the percent decrease in exon 21xinclusion at the indicated doses (n=4 per group). FIG. 15B depicts agraph plotting the percent increase in Scn1a mRNA at the indicated doses(n=4 per group).

FIG. 16 depicts a selected Scn1a targeting ASO administered at a bugdose via ICV injection in postnatal day 2 mice evaluated at day 5post-injection by Taqman qPCR of SCN1A, SCN2A, SCN3A, SCN4A, SCN5A,SCN7A, SCN8A, SCN9A, SCN10A, and SCN11A to assess target selectivity.Taqman-qPCR amplification results normalized to Gapdh, obtained usingEx20x+1 ASO, are plotted as fold change relative to PBS injected mice(n=3-6 per group).

FIGS. 17A-B depict exemplary results from intracerebroventricular (ICV)injection at postnatal day 2 of a selected ASO at the indicated dose inwild type (WT) or heterozygous Dravet mice (HET) F1 mice from129S-Scn1a^(tm1Kea)×C57BL/6J crosses at 3 days post-injection (n=9-14per group). FIG. 17A depicts a graph from results of a Taqman qPCR assayperformed using a probe spanning exons 21 and 22. The products werenormalized to Gapdh internal control and fold-change of ASO-injectedrelative to PBS-injected brains is plotted. FIG. 17B depicts a graphfrom results of a western blot performed using an anti-Nav1.1 antibody.The products were normalized to Ponceau-stained bands and fold-change ofASO-injected relative to PBS-injected brains is plotted.

FIG. 19 is a graph plotting increase in Scn1a mRNA level in coronalbrain slices of mice over the time post ICV injection of a SCN1Atargeting ASO. As depicted, increase in Scn1a mRNA level, as quantifiedby Taqman qPCR, was maintained for at least 80 days post-injection(n=3-9 per group).

FIG. 20 is an exemplary survival curve demonstrating 100% survivalbenefit provided by a SCN1A targeting ASO in Dravet mouse model. WT andheterozygous Dravet mice (+/−), F1 offspring from129S-scn1a^(tm1Kea)×C57BL/6J crosses, received a single dose ICVinjection of 20 μg PBS or ASO blindly (treatment marked as A or B) onpostnatal day 2, and their survival was monitored. As depicted, mice inA +/− group (Dravet mice receiving PBS treatment) started to die fromabout postnatal day 16, whereas all mice of other three groups,including B+/−(Drave mice receiving ASO treatment) group, survived atleast 35 days postnatal (n=32-39 per group).

FIG. 18 depicts exemplary results of a SCN1A exon 20x region ASOmicrowalk in RenCells via free uptake. ASOs were designed to coverregions around three previously identified targeting ASOs in FIG. 6(marked by stars) by shifting 1 nucleotides at a time (6-41) or bydecreasing the length of ASO 17 (1-5). The graph depicts the percentexon 20x inclusion as measured by SYBR-green qPCR. The black lineindicates no change with respect to no ASO (−).

Sequences of ASOs are summarized in Table 7a and Table 7b.

TABLE 7a  Sequences of ASOs targeting human SCN1A ASO ID Sequence 5′-3′SEQ ID NO: 1 TTGGAGCAAGATTATC 304 2 GTTGGAGCAAGATTATC 305 3GTTGGAGCAAGATTAT 306 4 AGTTGGAGCAAGATTAT 307 5 AGTTGGAGCAAGATTA 308 6GATTATCCTATACAAAAT 309 7 AGATTATCCTATACAAAA 310 8 AAGATTATCCTATACAAA 3119 CAAGATTATCCTATACAA 312 10 GCAAGATTATCCTATACA 313 11 AGCAAGATTATCCTATAC314 12 GAGCAAGATTATCCTATA 315 13 GGAGCAAGATTATCCTAT 316 14TGGAGCAAGATTATCCTA 317 15 GTTGGAGCAAGATTATCC 318 16 TTGGAGCAAGATTATCCT319 18 AAGTTGGAGCAAGATTAT 320 19 CAAGTTGGAGCAAGATTA 321 20CCAAGTTGGAGCAAGATT 322 21 TCCAAGTTGGAGCAAGAT 323 22 AGTACCCATAATAAAGGG324 23 AATACAGTACCCATAATA 325 24 ATTAAAGGTAGCAAAAGG 326 25GATTAAAGGTAGCAAAAG 327 26 GGATTAAAGGTAGCAAAA 328 27 AGGATTAAAGGTAGCAAA329 29 CAAGGATTAAAGGTAGCA 330 30 GCAAGGATTAAAGGTAGC 331 31TGCAAGGATTAAAGGTAG 332 32 GTGCAAGGATTAAAGGTA 333 33 AGTCACAGTGCAAGGATT334 34 AAGTCACAGTGCAAGGAT 335 35 TAAGTCACAGTGCAAGGA 336 36ATAAGTCACAGTGCAAGG 337 38 ACATAAGTCACAGTGCAA 338 39 CACATAAGTCACAGTGCA339 40 ACACATAAGTCACAGTGC 340 41 TACACATAAGTCACAGTG 341

TABLE 7b  Sequences of ASOs targeting human SCN1A ASO ID Sequence 5′-3′SEQ ID NO:  1_U UUGGAGCAAGAUUAUC 342  2_U GUUGGAGCAAGAUUAUC 343  3_UGUUGGAGCAAGAUUAU 344  4_U AGUUGGAGCAAGAUUAU 345  5_U AGUUGGAGCAAGAUUA346  6_U GAUUAUCCUAUACAAAAU 347  7_U AGAUUAUCCUAUACAAAA 348  8_UAAGAUUAUCCUAUACAAA 349  9_U CAAGAUUAUCCUAUACAA 350 10_UGCAAGAUUAUCCUAUACA 351 11_U AGCAAGAUUAUCCUAUAC 352 12_UGAGCAAGAUUAUCCUAUA 353 13_U GGAGCAAGAUUAUCCUAU 354 14_UUGGAGCAAGAUUAUCCUA 355 15_U GUUGGAGCAAGAUUAUCC 356 16_UUUGGAGCAAGAUUAUCCU 357 18_U AAGUUGGAGCAAGAUUAU 358 19_UCAAGUUGGAGCAAGAUUA 359 20_U CCAAGUUGGAGCAAGAUU 360 21_UUCCAAGUUGGAGCAAGAU 361 22_U AGUACCCAUAAUAAAGGG 362 23_UAAUACAGUACCCAUAAUA 363 24_U AUUAAAGGUAGCAAAAGG 364 25_UGAUUAAAGGUAGCAAAAG 365 26_U GGAUUAAAGGUAGCAAAA 366 27_UAGGAUUAAAGGUAGCAAA 367 29_U CAAGGAUUAAAGGUAGCA 368 30_UGCAAGGAUUAAAGGUAGC 369 31_U UGCAAGGAUUAAAGGUAG 370 32_UGUGCAAGGAUUAAAGGUA 371 33_U AGUCACAGUGCAAGGAUU 372 34_UAAGUCACAGUGCAAGGAU 373 35_U UAAGUCACAGUGCAAGGA 374 36_UAUAAGUCACAGUGCAAGG 375 38_U ACAUAAGUCACAGUGCAA 376 39_UCACAUAAGUCACAGUGCA 377 40_U ACACAUAAGUCACAGUGC 378 41_UUACACAUAAGUCACAGUG 379

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A pharmaceutical composition comprising anantisense oligomer that comprises a sequence selected from the groupconsisting of SEQ ID NOs: 21-61, 64-67, 210-250, 253-256 and 304-379,and a pharmaceutically acceptable excipient.
 2. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer comprises asequence selected from the group consisting of SEQ ID NOs: 21-61 and64-67.
 3. The pharmaceutical composition of claim 1, wherein theantisense oligomer comprises a sequence selected from the groupconsisting of SEQ ID NOs: 210-250 and 253-256.
 4. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer comprises asequence selected from the group consisting of SEQ ID NOs: 304-379. 5.The pharmaceutical composition of claim 1, wherein the antisenseoligomer is a sequence selected from the group consisting of SEQ ID NOs:21-61, 64-67, 210-250, 253-256, and 304-379.
 6. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer comprises a2′-O-methoxyethyl moiety.
 7. The pharmaceutical composition of claim 6,wherein each nucleotide of the antisense oligomer comprises a2′-O-methoxyethyl moiety.
 8. The pharmaceutical composition of claim 1,wherein the antisense oligomer consists of from 8 to 50 nucleobases. 9.The pharmaceutical composition of claim 1, wherein the antisenseoligomer consists of from 12 to 20 nucleobases.
 10. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition isformulated for intrathecal injection or intracerebroventricularinjection.
 11. The pharmaceutical composition of claim 1, wherein theantisense oligomer comprises a backbone modification, a modified sugarmoiety or a combination thereof.
 12. A composition comprising a viralvector encoding an antisense oligomer; wherein the antisense oligomerbinds to a targeted portion of a pre-mRNA that contains a non-sensemediated RNA decay-inducing exon (NMD exon mRNA) and that encodes Nav1.1protein, whereby the antisense oligomer promotes exclusion of the NMDexon from the NMD exon mRNA encoding Nav1.1.
 13. The composition ofclaim 12, wherein the antisense oligomer comprises a sequence selectedfrom the group consisting of SEQ ID NOs: 21-67, 210-256 and 304-379. 14.The composition of claim 12, wherein the antisense oligomer comprises asequence selected from the group consisting of SEQ ID NOs: 21-61, 64-67,210-250, 253-256, and 304-379.
 15. The composition of claim 12, whereinthe antisense oligomer comprises a sequence selected from the groupconsisting of SEQ ID NOs: 32-35, 39, 40, 42-45, 49-51, 54-59, 66,221-224, 228, 229, 231-234, 238-240, 243-248, 255, 304-308, 312-316,318-324, 326-335, 337-346, 350-354, 356-362, 364-373 and 375-379. 16.The pharmaceutical composition of claim 1, wherein the antisenseoligomer comprises a sequence selected from the group consisting of SEQID NOs: 32-35, 39, 40, 42-45, 49-51, 54-59, 66, 221-224, 228, 229,231-234, 238-240, 243-248, 255, 304-308, 312-316, 318-324, 326-335,337-346, 350-354, 356-362, 364-373 and 375-379.
 17. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 32or
 221. 18. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 33 or
 222. 19. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 42or
 231. 20. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 43 or
 232. 21. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 44or
 233. 22. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 51 or
 240. 23. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 54or
 243. 24. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 55 or
 244. 25. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 56or
 245. 26. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 57 or
 246. 27. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 58or
 247. 28. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 59 or
 248. 29. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 304or
 342. 30. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 305 or
 343. 31. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 306or
 344. 32. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 307 or
 345. 33. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 308or
 346. 34. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 312 or
 350. 35. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 313or
 351. 36. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 314 or
 352. 37. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 315or
 353. 38. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 316 or
 354. 39. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 318or
 356. 40. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 319 or
 357. 41. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 320or
 358. 42. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 321 or
 359. 43. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 322or
 360. 44. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 323 or
 361. 45. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 324or
 362. 46. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 326 or
 364. 47. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 327or
 365. 48. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 328 or
 366. 49. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 329or
 367. 50. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 330 or
 368. 51. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 331or
 369. 52. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 332 or
 370. 53. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 333or
 371. 54. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 334 or
 372. 55. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 335or
 373. 56. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 337 or
 375. 57. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 338or
 376. 58. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 339 or
 377. 59. The pharmaceuticalcomposition of claim 1, wherein the antisense oligomer is SEQ ID NO: 340or
 378. 60. The pharmaceutical composition of claim 1, wherein theantisense oligomer is SEQ ID NO: 341 or
 379. 61. The pharmaceuticalcomposition of any one of claims 16-60, wherein the antisense oligomercomprises a 2′-O-methoxyethyl moiety.
 62. The pharmaceutical compositionof any one of claims 16-60, wherein each nucleotide of the antisenseoligomer comprises a 2′-O-methoxyethyl moiety.